Gamma-AApeptides with potent and broad-spectrum antimicrobial activity

ABSTRACT

The present invention is directed to a novel class of antimicrobial agents called γ-AApeptides. The current invention provides various categories of γ-AApeptides, for example, linear γ-AApeptides, cyclic γ-AApeptides, and lipidated γ-AApeptides. γ-AApeptides of the current invention are designed to exert antimicrobial activity while being stable and non-toxic. γ-AApeptides also do not appear to lead to the development of microbial resistance in treated microorganisms. Thus, the disclosed γ-AApeptides can be used for the treatment of various medical conditions associated with pathogenic microorganisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/292,162, filed Oct. 13, 2016, now U.S. Pat. No. 10,144,764, which isa continuation of U.S. application Ser. No. 14/374,018, filed Jul. 23,2014, now U.S. Pat. No. 9,499,587, which is the U.S. national stageapplication of International Patent Application No. PCT/US2013/022695,filed Jan. 23, 2013, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/589,496, filed Jan. 23, 2012, the disclosures ofwhich are hereby incorporated by reference in their entirety, includingall figures, tables and nucleic acid sequences.

BACKGROUND OF THE INVENTION

Antimicrobial peptides are small cationic amphiphilic peptides found invirtually all living organisms (Marr et al., 2006). They play animportant role in innate immune defense against various infections(Hancock et al., 2006). In the last decade, there has been significantinterest in the development of antimicrobial peptides because of theemergence of antibiotic resistance (Marr et al., 2006; Hancock et al.,2006).

Compared to conventional antibiotics, which target specific metabolicprocesses in bacteria (Alekshun et al., 2007), antimicrobial peptidesare able to form amphipathic structures, where cationic and hydrophobicgroups are segregated into two regions, so as to facilitate interactionwith the negatively charged bacterial cytoplasmic membrane(Chongsiriwatana et al., 2008). Such interaction is based on the globalchemical properties of peptides, rather than their precise sequence(Scott et al., 2008). Therefore, antimicrobial peptides are unlikely tobe hindered by the resistance mechanisms observed for current antibiotictreatments (Marr et al., 2006). Furthermore, unlike conventionalantibiotics, antimicrobial peptides exhibit broad-spectrum activityagainst both Gram-positive and Gram-negative bacteria, and even fungiand viruses (Marr et al., 2006; Hancock et al., 2006). They appear to beideal antibiotic agents to supplement or replace existing treatments(Chongsiriwatana et al., 2008).

However, despite significant enthusiasm, there are intrinsic drawbacksassociated with the development of peptide antibiotics due to thepeptidic nature of antimicrobial peptides. These include potentialimmunoreactivity, susceptibility to enzymatic degradation, etc. (Zaiou,2007). Non-natural peptidomimetic approaches that mimic antimicrobialpeptides may circumvent these impediments by introducing amide bondisosteres, and modifying the peptide backbone so as to improveresistance to proteolytic hydrolysis (Violette et al., 2009). To thisend, non-natural antimicrobial oligomers, such as 0-peptides, peptoids,arylamides, and oilgourea, have been developed (Tew et al., 2009).However, their rational design sometimes turns out to be complicated dueto the difficulty of introducing a variety of functional groups tofine-tune their activity and selectivity, and the inconsistency of theirstructure-activity-relationship (Fowler et al., 2009). Furthermore,recent research findings from many groups suggest that helicalconformations, in which lipophilic and cationic side chains are globallysegregated, are not necessary for antimicrobial activity (Schmitt etal., 2007; Mowery et al., 2007). Indeed, a pre-organized secondarystructure seems unnecessary for bacterial killing (Scott et al., 2008);instead, oligomers with a strong propensity for helical conformation orconformational rigidity may lead to high hemolytic activity(Chongsiriwatana et al., 2008; Ivankin et al., 2010). Potentantimicrobial activity may actually require the presence of flexible oreven random coiled backbones, where side groups are segregated intohydrophobic and cationic regions upon interaction with bacterialmembranes (Scott et al., 2008; Ivankin et al., 2010), even if theamphiphilic conformation is irregular and non-helical.

There remains a need for the development of antimicrobial peptidemimetics suitable for the treatment of various microbial diseases. Thisapplication discloses a new class of antimicrobial peptidemimetics—γ-AApeptides developed by a simple design strategy. Certainγ-AApeptides were able to disrupt protein-protein interactions (Niu, Huet al., 2011) and recognize nucleic acids with high affinity andspecificity (Niu, Jones et al., 2011), and were highly resistant toprotease degradation (Niu, Hu et al., 2011). Moreover, the synthesis anddiversification of γ-AApeptides is efficient and straightforward (Niu,Hu et al., 2011), strengthening their potential to generate focusedlibraries for drug-lead screening. The antimicrobial γ-AApeptidesdisclosed herein are potent and have broad-spectrum activity, includingactivity against clinically-relevant strains that are unresponsive tomost antibiotics. Also, γ-AApeptides are not prone to select fordrug-resistant bacterial strains.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a novel class of antimicrobialagents called γ-AApeptides (γ-AApeptides include linear γ-AApeptides,cyclic γ-AApeptides, and lipidated γ-AApeptides), which are designed toexert antimicrobial activity while being stable, non-toxic and avoidingdevelopment of resistance to the γ-AApeptides. Thus, the disclosedγ-AApeptides can be used for the treatment of various medical conditionsassociated with pathogenic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1B. Illustration of antimicrobial γ-AApeptide design. FIG. 1A,basic representation of amphiphilic γ-AApeptide structure; FIG. 1B,conformational change of γ-AApeptide upon interaction with bacterialcell membranes.

FIG. 2. The structures of oligomers tested in Example 1 forantimicrobial activity. Underlined building blocks are hydrophobicbuilding blocks containing two hydrophobic side chains; the rest of thebuilding blocks in the sequences are amphiphilic with one cationic andone hydrophobic side chain.

FIG. 3. The structure of an α-peptide and the corresponding γ-AApeptideand γ-PNA.

FIGS. 4A and B. The development of resistance by S. aureus ATCC 33592towards γ5 and norfloxacin (FIG. 4A) and lipidated γAA2 and norfloxacin(FIG. 4B).

FIG. 5. Fluorescence micrographs of E. coli and B. subtilis treated with10 μg/ml γ-AApeptide γ5 for 2 h. a1-a4, E. coli. a1, control, notreatment, DAPI stained; a2, control, no treatment, PI stained; a3, γ5treatment, DAPI stained; a4, γ5 treatment, PI stained. b1-b4, B.subtilis. b1, control, no treatment, DAPI stained; b2, control, notreatment, PI stained; b3, γ5 treatment, DAPI stained; b4, γ5 treatment,PI stained. Scale bar: 2 μm for E. coli and for B. subtilis.

FIGS. 6A-6C. FIG. 6A shows the general synthesis of cyclic γ-AApeptidesvia on-resin cyclization. FIG. 6B shows the structures of cyclicγ-AApeptides. FIG. 6C shows the structure of linear γ-AApeptide γ5.

FIGS. 7A-7B. Illustration of cyclic antimicrobial γ-AApeptide design.FIG. 7A, basic representation of the amphiphilic γ-AApeptide buildingblock; FIG. 7B, amphipathic cyclic γ-AApeptide with globally amphipathicconformation.

FIG. 8. The energy-minimized structure of HW-B-13. The computer modelingwas carried out using ChemBioOffice MM2 energy minimization.

FIGS. 9A-9G. MTT cytotoxicity assay of N2a/APP cells treated withdifferent concentrations of cyclic γ-Aapeptides (HW-B-3 treatment, FIG.9A; HW-B-4 treatment, FIG. 9B; HW-B-5 treatment, FIG. 9C; HW-B-11treatment, FIG. 9D; HW-B-12 treatment, FIG. 9E; HW-B-13 treatment, FIG.9F; HW-B-14 treatment, FIG. 9G).

FIG. 10. Depolarization of the cytoplasmic membrane of S. aureus bycyclic γ-AApeptides.

FIG. 11. Fluorescence micrographs of B. subtilis treated with 5 μg/mlcyclic γ-AApeptide HW-B-13 for 2 h. a1, control, no treatment, DAPIstained; a2, control, no treatment, PI stained; a3, control, notreatment, the merged view. b1, HW-B-13 treatment, DAPI stained; b2,HW-B-13 treatment, PI stained; b3, HW-B-13 treatment, the merged view.Scale bar: 2 μm.

FIG. 12. γ-AApeptides used in antimicrobial assays. The first structureshows the general structure of γ-AApeptide building blocks.

FIG. 13. Fluorescence micrographs of E. coli and B. subtilis treatedwith 10 μg/mL of lipidated γ-AApeptide, Sequence 13, for 2 h: (a1-a4) E.coli; (a1) control, no treatment, DAPI stained; (a2) control, notreatment, PI stained; (a3) Sequence 13 treatment, DAPI stained; (a4)Sequence 13 treatment, PI stained; (b1-b4) B. subtilis; (b1) control, notreatment, DAPI stained; (b2) control, no treatment, PI stained; (b3)Sequence 13 treatment, DAPI stained; (b4) Sequence 13 treatment, PIstained.

FIG. 14. Fluorescence micrographs of E. coli and B. subtilis treatedwith 10 jag/ml lipidated γ-AApeptide, Sequence 6, for 2 h. a1-a4, E.coli. a1, control, no treatment, DAPI stained; a2, control, notreatment, PI stained; a3, Sequence 6 treatment, DAPI stained; a4,Sequence 6 treatment, PI stained. b1-b4, B. subtilis. b1, control, notreatment, DAPI stained; b2, control, no treatment, PI stained; b3,Sequence 6 treatment, DAPI stained; b4, Sequence 6 treatment, PIstained. Scale bar: 2 jam for both bacteria.

FIG. 15. Depolarization of the membrane of S. aureus. The fluorescenceintensity of membrane potential-sensitive dye DiSC3 was used as thepositive control.

FIG. 16. Development of resistance by S. aureus ATCC 33592 towardlipidated γ-AApeptide, Sequence 6, and norfloxacin.

FIG. 17. MTT assay of N2a/APP cells treated with differentconcentrations of lipidated γ-AApeptide, Sequence 6.

DETAILED DISCLOSURE OF THE INVENTION

The present invention is directed to a novel class of antimicrobialagents, γ-AApeptides, which include linear γ-AApeptides, cyclicγ-AApeptides, and lipidated γ-AApeptides. γ-AApeptides of the currentinvention are designed to exert antimicrobial activity while beingstable, non-toxic and avoiding development of resistance to theγ-AApeptides. The antimicrobial agents of the current invention aretermed “γ-AApeptides” (Niu, Hu et al., 2011) because they contain anN-acylated-N-aminoethyl amino acid unit (FIG. 3). In this unit, one sidechain is connected to the γ-C in relation to the carbonyl group, and theother side chain is linked to the central N through acylation. As shownin FIG. 3, γ-AApeptides are able to project an identical number of sidechains to natural peptides of the same length; therefore, they havegreat potential to be developed for peptide mimicry. Compared toconventional peptides, γ-AApeptides are much superior in both theirlimitless potential for diversification and their inherent resistance tobiodegradation. Thus, the disclosed γ-AApeptides can be used for thetreatment of various medical conditions associated with pathogenicmicroorganisms.

One aspect of the present invention relates to a novel class ofcompounds (γ-AApeptides: which include linear γ-AApeptides, cyclicγ-AApeptides, and lipidated γ-AApeptides), which are based on positivelycharged (cationic) and/or hydrophobic groups (also referred to herein assubunits). In one embodiment, γ-AApeptides can comprise at least 5 ormore cationic groups. Another embodiment provides γ-AApeptides thatcomprise both cationic groups and hydrophobic groups. In either of theseembodiments, the γ-AApeptides disclosed herein have antimicrobialactivity. γ-AApeptides disclosed herein can be between 5 and 50 subunitsin length, preferably at least seven subunits in length and no more than50 subunits in length.

Another aspect of the invention provides γ-AApeptides of the followingformulas:

wherein:

a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

d is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

f is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

g is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

h is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

G₁, G₂, G₃ and G₄ are, independently, hydrogen or a blocking group;

R₁ is a straight or branched chain C₁ to C₁₀ alkyl group (e.g., amethyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl,s-butyl or t-butyl group); —CH₂—CH₂—S—CH₃; a —(CH₂)₁₋₅-aryl group; or an—(CH₂)₁₋₅-heteroaryl group, wherein the alkyl group, the aryl group orthe heteroaryl group can be substituted or unsubstituted;

R₂ is an aryl group, a substituted aryl group, a heteroaryl group, or asubstituted heteroaryl group;

R₃ is an aryl group, a substituted aryl group, a heteroaryl group, or asubstituted heteroaryl group; and

R₄ is an aryl group, a substituted aryl group, a heteroaryl group, or asubstituted heteroaryl group.

In certain embodiments, R₁ is a methyl, ethyl, n-propyl, isopropyl,cyclopropyl, n-butyl, isobutyl, s-butyl or t-butyl group; R₂, R₃ and R₄are phenyl groups; g, h and i are, independently, 2-4 (preferably 2); eand f are, independently, 2-5 (preferably 4); and G₁, G₂, G₃ and G₄ are,independently, hydrogen or a blocking group. The compounds of formula Ican be linear, cyclic or lipidated (i.e., lipidated with a saturated orunsaturated lipid). γ-AApeptides can be lipidated at any reactive groupon the γ-AApeptide; however, in some embodiments the peptides arelipidated at the amino terminus of the γ-AApeptide (see, for example,FIG. 2). As noted above, the lipid can be saturated or unsaturated andcertain embodiments utilize a C₁₀-C₂₀ saturated or unsaturated lipidattached to the amino terminus of the γ-AApeptide. In various otherembodiments, C₁₆-C₁₈ saturated or unsaturated lipids (e.g., palmitic oroleic acids) can be used.

The term “alkyl” refers to a straight, branched or cyclic chainhydrocarbon radical with only single carbon-carbon bonds. Representativeexamples include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, andcyclohexyl, all of which may be optionally substituted. Alkyl groups areC₁-C₁₂ and include alkyl groups that are C₁-C₈ in some embodiments.

The term “aryl” refers to aromatic groups which have 5-14 ring atoms andat least one ring having a conjugated pi electron system and includescarbocyclic aryl, heterocyclic aryl and biaryl groups, all of which maybe optionally substituted. Carbocyclic aryl groups are groups whichhave, in various embodiments, 6-10 or 6-14 ring atoms wherein the ringatoms on the aromatic ring are carbon atoms. Carbocyclic aryl groupsinclude monocyclic carbocyclic aryl groups and polycyclic or fusedcompounds such as optionally substituted naphthyl groups. Heterocyclicaryl or heteroaryl groups are groups which have, in various embodiments,5-10 or 5-14 ring atoms wherein 1 to 4 heteroatoms are ring atoms in thearomatic ring and the remainder of the ring atoms being carbon atoms.Suitable heteroatoms include oxygen, sulfur, nitrogen, and selenium.Suitable heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl,N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl,imidazolyl, and the like, all optionally substituted. The term “biaryl”represents aryl groups which have 5-14 atoms containing more than onearomatic ring including both fused ring systems and aryl groupssubstituted with other aryl groups. Such groups may be optionallysubstituted. Suitable biaryl groups include naphthyl and biphenyl.“Substituted aryl” and “substituted heteroaryl” refer to aryl andheteroaryl groups substituted with 1-3 substituents. These substituentsare selected from the group consisting of lower alkyl, lower alkoxy,lower perhaloalkyl, halo, hydroxy, and amino.

The term “lower”, referred to herein in connection with organic radicalsor compounds respectively, defines such as with up to and including 10,in one aspect up to and including 6, and in another aspect one to fourcarbon atoms. Such groups may be straight chain, branched, or cyclic.

The term “alkoxy” refers to the group alkyl —O—.

The term “perhalo” refers to groups wherein every C—H bond has beenreplaced with a C-halo bond on an aliphatic or aryl group. Suitableperhaloalkyl groups include —CF₃ and —CFCl₂.

The term “halogen” or “halo” refers to —F, —Cl, —Br and —I.

The terms “heterocyclic”, “heterocyclic alkyl” or “heterocycloalkyl”refer to cyclic groups of 3 to 10 atoms, and in one aspect are 3 to 6atoms, containing at least one heteroatom, in a further aspect 1 to 3heteroatoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen.Heterocyclic groups may be attached through a nitrogen or through acarbon atom in the ring. The heterocyclic alkyl groups includeunsaturated cyclic, fused cyclic and spirocyclic groups. Suitableheterocyclic groups include pyrrolidinyl, morpholino, morpholinoethyl,and pyridyl.

In certain embodiments, R1 can be a group selected from:

Two blocking (protecting) groups (t-Boc, Fmoc) are commonly used insolid-phase peptide synthesis. The t-Boc blocking group(tert-butyloxycarbonyl) is used to temporarily protect an α-amino group.Briefly, the t-Boc group is covalently bound to the amino group tosuppress its nucleophilicity and the C-terminal amino acid is covalentlylinked to the resin through a linker. The t-Boc group is removed withacid, such as trifluoroacetic acid (TFA). This forms apositively-charged amino group, which is simultaneously neutralized andcoupled to an incoming activated amino acid. The Fmoc protecting group(9-fluorenylmethyloxycarbonyl; Fmoc) can also be used and provides for amilder deprotection scheme. This method utilizes a base, usuallypiperidine in DMF, in order to remove the Fmoc group to expose theα-amino group for reaction with an incoming activated amino acid.Peptide synthesis using an Fmoc protecting group uses a base to removethe Fmoc group and provides for a neutral exposed amine group.

As used herein the term “antimicrobial activity” refers to the abilityof a γ-AApeptide to kill or reduce the viability of a pathogenicmicroorganism selected from the group consisting of a prokaryoticorganism, a eubacterium, an archaebacterium, a yeast, a fungus, an alga,a protozoan and a parasite.

The term γ-AApeptides includes linear γ-AApeptides, cyclic γ-AApeptides,and lipidated γ-AApeptides (also called lipo-γ-AApeptides), unlessotherwise specified.

Another aspect of the invention provides cyclic γ-AApeptides. Theinvention also provides design and synthesis of cyclic γ-AApeptides. Inan embodiment of the invention, the cyclic γ-AApeptides are synthesizedby on-resin cyclization. The facile synthesis of cyclic γ-AApeptides mayfurther expand the applications of γ-AApeptides in biomedical sciences.Cyclic peptidomimetics are expected to have improved antimicrobialactivity due to constraints induced by cyclization. These cyclicγ-AApeptides are potent and active against fungi and Gram-positive andGram-negative bacterial pathogens.

A further aspect of the invention provides lipidated γ-AApeptides.Lipidated γ-AApeptides of the current invention are produced byattaching lipid molecules to γ-AApeptides of the current invention.Lipidated γ-AApeptides, containing hydrophobic alkyl tails and shortcationic γ-AApeptide sequences, display more potent, broad-spectrum, andhighly selective antimicrobial activities against fungi and a series ofclinically relevant Gram-positive and Gram-negative bacteria.Additionally, lipidated γ-AApeptides do not elicit drug resistance, forexample, in S. aureus, even after 17 rounds of passaging.

In another aspect of the invention, compositions comprising aγ-AApeptide as disclosed herein are provided. In this aspect of theinvention, one or more a γ-AApeptides are formulated in apharmaceutically acceptable excipient or diluent suitable foradministration to a subject. Techniques for formulation andadministration of drugs may be found in “Remington's PharmaceuticalSciences”, Mack Publishing Co., Easton, Pa., latest edition, which isincorporated herein by reference.

The compositions described herein can also further comprise anadditional therapeutically active agent (e.g., an antimicrobial agent oran antibiotic). Non-limiting examples of antimicrobial and antibioticagents that are suitable for use in this context of the presentinvention include: mandelic acid, 2,4-dichlorobenzenemethanol,4-[bis(ethylthio)methyl]-2-methoxyphenol, 4-epi-tetracycline,4-hexylresorcinol, 5,12-dihydro-5,7,12,14-tetrazapentacen,5-chlorocarvacrol, 8-hydroxyquinoline, acetarsol, acetylkitasamycin,acriflavin, alatrofloxacin, ambazon, amfomycin, amikacin, amikacinsulfate, aminoacridine, aminosalicylate calcium, aminosalicylate sodium,aminosalicylic acid, ammoniumsulfobituminat, amorolfin, amoxicillin,amoxicillin sodium, amoxicillin trihydrate, amoxicillin-potassiumclavulanate combination, amphotericin B, ampicillin, ampicillin sodium,ampicillin trihydrate, ampicillin-sulbactam, apalcillin, arbekacin,aspoxicillin, astromicin, astromicin sulfate, azanidazole,azidamfenicol, azidocillin, azithromycin, azlocillin, aztreonam,bacampicillin, bacitracin, bacitracin zinc, bekanamycin, benzalkonium,benzethonium chloride, benzoxonium chloride, berberine hydrochloride,biapenem, bibrocathol, biclotymol, bifonazole, bismuth subsalicylate,bleomycin antibiotic complex, bleomycin hydrochloride, bleomycinsulfate, brodimoprim, bromochlorosalicylanilide, bronopol,broxyquinolin, butenafine, butenafine hydrochloride, butoconazol,calcium undecylenate, candicidin antibiotic complex, capreomycin,carbenicillin, carbenicillin disodium, carfecillin, carindacillin,carumonam, carzinophilin, caspofungin acetate, cefacetril, cefaclor,cefadroxil, cefalexin, cefalexin hydrochloride, cefalexin sodium,cefaloglycin, cefaloridine, cefalotin, cefalotin sodium, cefamandole,cefamandole nafate, cefamandole sodium, cefapirin, cefapirin sodium,cefatrizine, cefatrizine propylene glycol, cefazedone, cefazedone sodiumsalt, cefazolin, cefazolin sodium, cefbuperazone, cefbuperazone sodium,cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren,cefditoren pivoxil, cefepime, cefepime hydrochloride, cefetamet,cefetamet pivoxil, cefixime, cefmenoxime, cefmetazole, cefmetazolesodium, cefminox, cefminox sodium, cefmolexin, cefodizime, cefodizimesodium, cefonicid, cefonicid sodium, cefoperazone, cefoperazone sodium,ceforanide, cefoselis sulfate, cefotaxime, cefotaxime sodium, cefotetan,cefotetan disodium, cefotiam, cefotiam hexetil hydrochloride, cefotiamhydrochloride, cefoxitin, cefoxitin sodium, cefozopran hydrochloride,cefpiramide, cefpiramide sodium, cefpirome, cefpirome sulfate,cefpodoxime, cefpodoxime proxetil, cefprozil, cefquinome, cefradine,cefroxadine, cefsulodin, ceftazidime, cefteram, cefteram pivoxil,ceftezole, ceftibuten, ceftizoxime, ceftizoxime sodium, ceftriaxone,ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime sodium,cetalkonium chloride, cetrimide, cetrimonium, cetylpyridinium,chloramine T, chloramphenicol, chloramphenicol palmitate,chloramphenicol succinate sodium, chlorhexidine, chlormidazole,chlormidazole hydrochloride, chloroxylenol, chlorphenesin,chlorquinaldol, chlortetracycline, chlortetracycline hydrochloride,ciclacillin, ciclopirox, cinoxacin, ciprofloxacin, ciprofloxacinhydrochloride, citric acid, clarithromycin, clavulanate potassium,clavulanate sodium, clavulanic acid, clindamycin, clindamycinhydrochloride, clindamycin palmitate hydrochloride, clindamycinphosphate, clioquinol, cloconazole, cloconazole monohydrochloride,clofazimine, clofoctol, clometocillin, clomocycline, clotrimazol,cloxacillin, cloxacillin sodium, colistin, colistin sodiummethanesulfonate, colistin sulfate, cycloserine, dactinomycin,danofloxacin, dapsone, daptomycin, daunorubicin, DDT, demeclocycline,demeclocycline hydrochloride, dequalinium, dibekacin, dibekacin sulfate,dibrompropamidine, dichlorophene, dicloxacillin, dicloxacillin sodium,didecyldimethylammonium chloride, dihydrostreptomycin,dihydrostreptomycin sulfate, diiodohydroxyquinolin, dimetridazole,dipyrithione, dirithromycin, DL-menthol, D-menthol,dodecyltriphenylphosphonium bromide, doxorubicin, doxorubicinhydrochloride, doxycycline, doxycycline hydrochloride, econazole,econazole nitrate, enilconazole, enoxacin, enrofloxacin, eosine,epicillin, ertapenem sodium, erythromycin, erythromycin estolate,erythromycin ethyl succinate, erythromycin lactobionate, erythromycinstearate, ethacridine, ethacridine lactate, ethambutol, ethanoic acid,ethionamide, ethyl alcohol, eugenol, exalamide, faropenem,fenticonazole, fenticonazole nitrate, fezatione, fleroxacin, flomoxef,flomoxef sodium, florfenicol, flucloxacillin, flucloxacillin magnesium,flucloxacillin sodium, fluconazole, flucytosine, flumequine,flurithromycin, flutrimazole, fosfomycin, fosfomycin calcium, fosfomycinsodium, framycetin, framycetin sulphate, furagin, furazolidone,fusafungin, fusidic acid, fusidic acid sodium salt, gatifloxacin,gemifloxacin, gentamicin antibiotic complex, gentamicin C1A, gentamycinsulfate, glutaraldehyde, gramicidin, grepafloxacin, griseofulvin,halazon, haloprogine, hetacillin, hetacillin potassium, hexachlorophene,hexamidine, hexetidine, hydrargaphene, hydroquinone, hygromycin,imipenem, isepamicin, isepamicin sulfate, isoconazole, isoconazolenitrate, isoniazid, isopropanol, itraconazole, josamycin, josamycinpropionate, kanamycin, kanamycin sulphate, ketoconazole, kitasamycin,lactic acid, lanoconazole, lenampicillin, leucomycin A1, leucomycin A13,leucomycin A4, leucomycin A5, leucomycin A6, leucomycin A7, leucomycinA8, leucomycin A9, levofloxacin, lincomycin, lincomycin hydrochloride,linezolid, liranaftate, 1-menthol, lomefloxacin, lomefloxacinhydrochloride, loracarbef, lymecyclin, lysozyme, mafenide acetate,magnesium monoperoxophthalate hexahydrate, mecetronium ethylsulfate,mecillinam, meclocycline, meclocycline sulfosalicylate, mepartricin,merbromin, meropenem, metalkonium chloride, metampicillin, methacycline,methenamin, methyl salicylate, methylbenzethonium chloride,methylrosanilinium chloride, meticillin, meticillin sodium,metronidazole, metronidazole benzoate, mezlocillin, mezlocillin sodium,miconazole, miconazole nitrate, micronomicin, micronomicin sulfate,midecamycin, minocycline, minocycline hydrochloride, miocamycin,miristalkonium chloride, mitomycin C, monensin, monensin sodium,morinamide, moxalactam, moxalactam disodium, moxifloxacin, mupirocin,mupirocin calcium, nadifloxacin, nafcillin, nafcillin sodium, naftifine,nalidixic acid, natamycin, neomycin A, neomycin antibiotic complex,neomycin C, neomycin sulfate, neticonazole, netilmicin, netilmicinsulfate, nifuratel, nifuroxazide, nifurtoinol, nifurzide, nimorazole,niridazole, nitrofurantoin, nitrofurazone, nitroxolin, norfloxacin,novobiocin, nystatin antibiotic complex, octenidine, ofloxacin,oleandomycin, omoconazol, orbifloxacin, ornidazole, ortho-phenylphenol,oxacillin, oxacillin sodium, oxiconazole, oxiconazole nitrate, oxoferin,oxolinic acid, oxychlorosene, oxytetracycline, oxytetracycline calcium,oxytetracycline hydrochloride, panipenem, paromomycin, paromomycinsulfate, pazufloxacine, pefloxacin, pefloxacin mesylate, penamecillin,penicillin G, penicillin G potassium, penicillin G sodium, penicillin V,penicillin V calcium, penicillin V potassium, pentamidine, pentamidinediisetionate, pentamidine mesilas, pentamycin, phenethicillin, phenol,phenoxyethanol, phenylmercuriborat, PHMB, phthalylsulfathiazole,picloxydin, pipemidic acid, piperacillin, piperacillin sodium,pipercillin sodium-tazobactam sodium, piromidic acid, pivampicillin,pivcefalexin, pivmecillinam, pivmecillinam hydrochloride, policresulen,polymyxin antibiotic complex, polymyxin B, polymyxin B sulfate,polymyxin B1, polynoxylin, povidone-iodine, propamidin, propenidazole,propicillin, propicillin potassium, propionic acid, prothionamide,protiofate, pyrazinamide, pyrimethamine, pyrithion, pyrrolnitrin,quinoline, quinupristin-dalfopristin, resorcinol, ribostamycin,ribostamycin sulfate, rifabutin, rifampicin, rifamycin, rifapentine,rifaximin, ritiometan, rokitamycin, rolitetracycline, rosoxacin,roxithromycin, rufloxacin, salicylic acid, secnidazol, seleniumdisulphide, sertaconazole, sertaconazole nitrate, siccanin, sisomicin,sisomicin sulfate, sodium thiosulfate, sparfloxacin, spectinomycin,spectinomycin hydrochloride, spiramycin antibiotic complex, spiramycinB, streptomycin, streptomycin sulphate, succinylsulfathiazole,sulbactam, sulbactam sodium, sulbenicillin disodium, sulbentin,sulconazole, sulconazole nitrate, sulfabenzamide, sulfacarbamide,sulfacetamide, sulfacetamide sodium, sulfachlorpyridazine, sulfadiazine,sulfadiazine silver, sulfadiazine sodium, sulfadicramide,sulfadimethoxine, sulfadoxine, sulfaguanidine, sulfalene, sulfamazone,sulfamerazine, sulfamethazine, sulfamethazine sodium, sulfamethizole,sulfamethoxazole, sulfamethoxazol-trimethoprim, sulfamethoxypyridazine,sulfamonomethoxine, sulfamoxol, sulfanilamide, sulfaperine,sulfaphenazol, sulfapyridine, sulfaquinoxaline, sulfasuccinamide,sulfathiazole, sulfathiourea, sulfatolamide, sulfatriazin,sulfisomidine, sulfisoxazole, sulfisoxazole acetyl, sulfonamides,sultamicillin, sultamicillin tosilate, tacrolimus, talampicillinhydrochloride, teicoplanin A2 complex, teicoplanin A2-1, teicoplaninA2-2, teicoplanin A2-3, teicoplanin A2-4, teicoplanin A2-5, teicoplaninA3, teicoplanin antibiotic complex, telithromycin, temafloxacin,temocillin, tenoic acid, terbinafine, terconazole, terizidone,tetracycline, tetracycline hydrochloride, tetracycline metaphosphate,tetramethylthiuram monosulfide, tetroxoprim, thiabendazole,thiamphenicol, thiaphenicol glycinate hydrochloride, thiomersal, thiram,thymol, tibezonium iodide, ticarcillin, ticarcillin-clavulanic acidmixture, ticarcillin disodium, ticarcillin monosodium, tilbroquinol,tilmicosin, tinidazole, tioconazole, tobramycin, tobramycin sulfate,tolciclate, tolindate, tolnaftate, toloconium metilsulfat, toltrazuril,tosufloxacin, triclocarban, triclosan, trimethoprim, trimethoprimsulfate, triphenylstibinsulfide, troleandomycin, trovafloxacin, tylosin,tyrothricin, undecoylium chloride, undecylenic acid, vancomycin,vancomycin hydrochloride, viomycin, virginiamycin antibiotic complex,voriconazol, xantocillin, xibornol, zinc undecylenate and variouscombinations thereof.

The γ-AApeptides described herein can be used for the treatment ofpathogenic microorganism infections. The option to include an additionaltherapeutically active agent may thus act synergistically againstvarious bacteria, fungi and other microorganisms. The phrase “pathogenicmicroorganism” is used to describe any microorganism which can cause adisease or disorder in subjects/mammals in general, particularly inhumans. The pathogenic microorganism may belong to any family oforganisms such as, but not limited to, prokaryotic organisms,eubacteria, archaebacteria, eukaryotic organisms, yeast, fungi, algae,protozoans, and other parasites. Non-limiting examples of pathogenicmicroorganisms are Plasmodium falciparum and related malaria-causingprotozoan parasites, Acanthamoeba and other free-living amoebae,Aeromonas hydrophila, Anisakis and related worms, Acinetobacterbaumanii, Ascaris lumbricoides, Bacillus cereus, Brevundimonas diminuta,Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens,Cryptosporidium parvum, Cyclospora cayetanensis, Diphyllobothrium,Entamoeba histolytica, certain strains of Escherichia coli,Eustrongylides, Giardia lamblia, Klebsiella pneumoniae, Listeriamonocytogenes, Nanophyetus, Plesiomonas shigelloides, Proteus mirabilis,Pseudomonas aeruginosa, Salmonella, Serratia odorifera, Shigella,Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus,Trichuris trichiura, Vibrio cholerae, Vibrio parahaemolyticus, Vibriovulnificus and other vibrios, Yersinia enterocolitica, Yersiniapseudotuberculosis and Yersinia kristensenii.

As used herein, the terms “treating” and “treatment” include abrogating,substantially inhibiting, slowing or reversing the progression of acondition, substantially ameliorating clinical or aesthetical symptomsof a condition or substantially preventing the appearance of clinical oraesthetic symptoms associated with a condition. As used herein, thephrase “therapeutically effective amount” describes an amount of thecomposition being administered which will relieve to some extent one ormore of the symptoms of the condition being treated. In some aspects ofthe invention, the phrase “therapeutically effective amount” refers to areduction in the viability of a microorganism to be treated with thedisclosed γ-AApeptides in vivo.

γ-AApeptides disclosed herein are antimicrobial agents which do notappear to induce resistance. The possible development of resistance toγ-AApeptides disclosed herein was assessed as discussed in the Examples.The results obtained in the antimicrobial-resistance studies showed thatexposing bacteria to the disclosed γ-AApeptides did not result indevelopment of resistance. γ-AApeptides disclosed herein display low ornegligible hemolytic activity in the systems tested.

Diseases capable of being treated in accordance with the disclosedinvention include, for example, actinomycosis, anthrax, aspergillosis,bacteremia, bacterial skin diseases, Bartonella infections, botulism,brucellosis, Burkholderia infections, Campylobacter infections,candidiasis, cat-scratch disease, Chlamydia infections, cholera,Clostridium infections, coccidioidomycosis, cryptococcosis,dermatomycoses, diphtheria, ehrlichiosis, typhus, Escherichia coliinfections, Fusobacterium infections, gangrene, general infections,general mycoses, gonorrhea, Gram-negative bacterial infections,Gram-positive bacterial infections, histoplasmosis, impetigo, Klebsiellainfections, legionellosis, leprosy, leptospirosis, Listeria infections,Lyme disease, malaria, maduromycosis, melioidosis, Mycobacteriuminfections, Mycoplasma infections, necrotizing fasciitis, Nocardiainfections, onychomycosis, ornithosis, pneumococcal infections,pneumonia, Pseudomonas infections, Q fever, rat-bite fever, relapsingfever, rheumatic fever, Rickettsia infections, Rocky Mountain spottedfever, Salmonella infections, scarlet fever, scrub typhus, sepsis,sexually transmitted bacterial diseases, staphylococcal infections,streptococcal infections, surgical site infections, tetanus, tick-bornediseases, tuberculosis, tularemia, typhoid fever, urinary tractinfections, Vibrio infections, yaws, Yersinia infections, Yersiniapestis infections, zoonoses and zygomycosis.

The γ-AApeptides disclosed herein have been shown to have high andselective affinity toward membranes of microorganisms. This feature canbe used to couple a γ-AApeptides disclosed herein to a linker and/orlabeling agent and used for the detection of infections in asubject/mammal, such as a rodent or a human. Accordingly, another aspectof the present invention provides an imaging probe for detecting apathogenic microorganism, the imaging probe comprising a γ-AApeptide andat least one labeling agent attached thereto (preferably through alinker). As is apparent to one skilled in the art, commerciallyavailable linkers reactive with free amine groups (primary or secondary)can be coupled to γ-AApeptides disclosed herein. Likewise, commerciallyavailable labeling agents, such as fluorescent probes (e.g., IRDYE 800CWNHS ester) or chelating agents (e.g., DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-NHS ester orNOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)-NHS ester), can alsobe linked to γ-AApeptides via primary or secondary amine groups.Non-limiting examples of commercially available linkers that can becoupled to γ-AApeptides disclosed herein include:

Diseases/infections capable of being diagnosed or identified inaccordance with the disclosed invention in a subject/mammal (such as arodent or a human) include, for example, actinomycosis, anthrax,aspergillosis, bacteremia, bacterial skin diseases, Bartonellainfections, botulism, brucellosis, Burkholderia infections,Campylobacter infections, candidiasis, cat-scratch disease, Chlamydiainfections, cholera, Clostridium infections, coccidioidomycosis,cryptococcosis, dermatomycoses, diphtheria, ehrlichiosis, typhus,Escherichia coli infections, Fusobacterium infections, gangrene, generalinfections, general mycoses, gonorrhea, Gram-negative bacterialinfections, Gram-positive bacterial infections, histoplasmosis,impetigo, Klebsiella infections, legionellosis, leprosy, leptospirosis,Listeria infections, Lyme disease, malaria, maduromycosis, melioidosis,Mycobacterium infections, Mycoplasma infections, necrotizing fasciitis,Nocardia infections, onychomycosis, ornithosis, pneumococcal infections,pneumonia, Pseudomonas infections, Q fever, rat-bite fever, relapsingfever, rheumatic fever, Rickettsia infections, Rocky Mountain spottedfever, Salmonella infections, scarlet fever, scrub typhus, sepsis,sexually transmitted bacterial diseases, staphylococcal infections,streptococcal infections, surgical site infections, tetanus, tick-bornediseases, tuberculosis, tularemia, typhoid fever, urinary tractinfections, Vibrio infections, yaws, Yersinia infections, Yersiniapestis infections, zoonoses and zygomycosis.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Design and Activity of γ-AApeptides

We prepared γ-AApeptide sequences (depicted in FIG. 2) and tested theirantimicrobial activity against a range of clinically-relevantGram-negative and Gram-positive bacteria, as well as the fungus C.albicans. γ1, γ2, γ3 are γ-AApeptides of different lengths that arecomposed of amphiphilic γ-AApeptide building blocks. γ4 contains onehydrophobic building block while γ5 contains two hydrophobic buildingblocks, an attempt to tune overall hydrophobicity of γ-AApeptides. Ascontrols, we included AMP magainin II (ml) (Padhee et al., 2011), a14-mer conventional peptide p1 (Padhee et al., 2011) with alternativephenylalanine and lysine residues, and the most potent antimicrobialα-AApeptide α1 reported by us recently (Padhee et al., 2011). Theantimicrobial activity of these oligomers was tested and listed inTable 1. Their hemolytic activity was also tested to evaluate theirselectivity.

A few sequences show very potent broad-spectrum activities against fungiand a series of clinically-relevant Gram-positive and Gram-negativebacteria, including pathogens that are unresponsive to most antibiotics(Table 1). The control sequence p1, which contains alternative phe andlys residues, the functional groups identical to γ3, does not show anyantimicrobial activity at all. Such an observation can be explained bythe peptide's intrinsic folding propensity (Padhee et al., 2011).Meanwhile, longer sequences are more potent and exhibit broad-spectrumactivity, compared to shorter sequences with similar structures, as seenfor the activities of γ1-γ3. This seems to indicate that the number ofcationic charges is important for interaction with bacterial membranes.Notably, γ3 is more potent and has a broader spectrum of activity ascompared to α3 (which has the same functional groups).

TABLE 1 The antimicrobial and hemolytic activities of oligomers.Magainin II is a natural amphipathic antimicrobial peptide used as thecomparison here. The sequences showing broad-spectrum antimicrobialactivity (γ3-γ5 and α1) are designated with an *. MIC (μg/ml) Organismγ1 γ2 γ3* γ4* γ5* pl α1* Ml Gram-negative E. coli >100 25 2.5-5  2.5-5   2.5-5   >100 4.5 40 K. pneumoniae >100 >100 >100 >1005 >100 >100 >100 (causing pneumonia) Gram-Positive B. subtilis >100 52.5 2.5 2 >100 2 40 S. epidermidis >100 >100 6.25-12.5  3.1-6.25 3.1-6.25 >100 10 >100 (multi-drug resistant) E. faecalis >100 >10012.5-25   12.5-25    3.1-6.25 >100 75 75 (vancomycin- resistant) S.aureus >100 >100 12.5-25    3.1-6.25 5 >100 75 >100 (methicillin-resistant) MRSA USA100 — — — — 5 — >100 >100 (leading hospital-associated strain, unresponsive to almost all antibiotics) B. anthracis— — 25-50 25-50 5 — >100 >100 (lethal bioweapon) Fungi >100 >100 C.albicans >500 >500 12.5-25   12.5-25    5-10 >100 20-30 75 Hemolysis(H₅₀) >500 >500 300 >500 >500 >500The further development of antimicrobial γ-AApeptides based on the leadγ3 yielded more potent sequences. As an initial attempt, we introduced asimple hydrophobic building block to tune the overall hydrophobicity andhydrophilicity of γ3, in order to evaluate the feasibility to tune theantimicrobial activity of γ-AApeptides. Such an effort led to thediscovery of γ5, in which two amphiphilic building blocks are replacedwith hydrophobic building blocks (containing two hydrophobic sidechains) compared to γ3, and only one side chain difference from γ4.While γ4 are not active against Gram-negative K. pneumoniae, γ5 potentlyinhibited the growth of all tested Gram-negative and Gram-positivebacteria, and also the fungus C. albicans. In fact, γ5 is amongst themost potent and broad-spectrum antimicrobial peptidomimetics reported todate. Most significantly, it arrested the growth of the USA100 lineageMRSA strain with extreme potency. This observation is of particularimportance as this strain displays resistance to a wealth of existingantimicrobial agents, and is broadly multi-drug resistant. As such, thismay satisfy urgent needs in hospitals for new therapeutics, since thisstrain has been identified as the most prevalent cause ofhospital-associated infections in the United States, and almost nocurrent antibiotics can effectively target it. γ5 also potently inhibitsB. anthracis, which is known to cause the highly lethal conditionanthrax. All tested γ-AApeptides have excellent selectivity for bacteriaand spare human blood cells, since the majority of them have H₅₀ of morethan 500 μg/ml; even the most hemolytic sequence γ5 still has aselectivity of at least 60-fold.

One of the biggest challenges for conventional antibiotics is theirsusceptibility to the development of resistance, which quickly abolishestheir efficacy. This situation has become more severe in recent years,and may lead to outbreaks of deadly infectious diseases that areuntreatable. To investigate the potential for bacteria to developresistance against the treatment of γ-AApeptides, meticillin-resistantS. aureus (ATCC 33592) was serially passaged in half-MIC concentrationsof γ5 and lipidated γAA2 (see Example 2), and new MIC values weredetermined every 24 h. As a positive control, parallel cultures wereexposed to serial 2-fold dilutions of the antibiotic norfloxacin (Choiet al., 2009). After 17 days, virtually no change in the MIC occurredfor γ5 over the 17 passages, whereas the MIC for norfloxacin started toincrease after just three passages, and the organism developed profoundresistance after 17 passages (MIC increased >20-fold). These resultsdemonstrate that MRSA strains do not readily develop resistance toγ-AApeptide γ5 or lipidated γAA2 (see FIGS. 4A and B).

In summary, we reported the identification of a new class ofantimicrobial peptidomimetics, γ-AApeptides, with potency andbroad-spectrum activity far superior to previous reported α-peptides(Padhee et al., 2011). These γ-AApeptides likely inhibit bacterialgrowth by mimicking conventional antimicrobial peptides through membranedisruption. This is an important observation as it suggests thepotential for virtually no development of resistance towards theseagents. Additionally, our approach to the development of antimicrobialpeptides avoids tedious and sometimes ineffective secondary structuredevelopment as has been observed in the design of other classes ofantimicrobial peptidomimetics.

1. General Experimental Methods for Example 1

α-amino acid esters and Knorr resin (0.66 mmol/g, 200-400 mesh) wereprovided by Chem-Impex International, Inc. All other reagents andsolvents were purchased from either Sigma-Aldrich or Fisher Scientific.α-peptides m1 and p1 were purchased from USF peptide facility and usedwithout further purification. γ-AApeptide building blocks weresynthesized following the previously reported procedure (Niu, Hu et al.,2011). NMR spectra of γ-AApeptide building blocks were obtained on aVarian Inova 400 instrument. Linear γ-AApeptides were prepared on Knorrresin in peptide synthesis vessels on a Burrell Wrist-Action shaker. Thelinear γ-AApeptides were analyzed and purified on an analytical and apreparative Waters HPLC system, respectively, and then dried on aLabcono lyophilizer. Molecular weights of γ-AApeptides were identifiedon a Bruker AutoFlex MALDI-TOF mass spectrometer.

2. Synthesis (Niu, Hu et al., 2011) and Characterization of γ-AApeptideBuilding Blocks

γ-AApeptide building blocks were synthesized following the previouslyreported procedure (Niu, Hu et al., 2011, the disclosures of which arehereby incorporated by reference in their entireties).

Compound 1. Yield 65% (two steps). ¹H NMR (DMSO-d₆, 400 MHz) δ=7.84 (d,J=7.2 Hz, 2H), 7.65-7.60 (m, 2H), 7.37 (t, J=7.6 Hz, 2H), 7.27 (m, 2H),7.22-7.08 (m, 5H), 6.71 (b, 1H), 4.28-4.02 (m, 3H), 3.98-3.57 (m, 2H),3.47-3.44 (m, 1H), 3.45 (dd, J=5.2, 13.2 Hz, 1H), 3.04-2.99 (m, 1H),2.85-2.84 (m, 2H), 2.73-2.53 (m, 3H), 2.47-2.31 (m, 1H), 1.33-1.11 (m,15H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.7, 172.3, 156.5, 156.4, 156.0,144.4, 144.3, 144.28, 144.2, 141.9, 141.8, 141.2, 128.7, 128.61, 128.59,128.0, 127.5, 127.4, 126.2, 126.1, 125.62, 125.58, 125.5, 120.5, 77.7,65.7, 65.6, 52.5, 51.1, 50.2, 51.1, 50.11, 50.09, 48.3, 47.3, 41.1,34.5, 34.0, 32.1, 31.7, 31.2, 31.0, 29.8, 29.7, 28.7, 23.7, 23.3, 23.27.HR-ESI: [M+H]⁺ cacl: 644.3330, found: 644.3338.

Compound 2. Yield 53% (two steps). ¹H NMR (DMSO-d₆, 400 MHz) δ=7.83 (d,J=8.0 Hz, 2H), 7.65-7.59 (m, 2H), 7.38-7.07 (m, 10H), 4.29-4.21 (m, 2H),4.17-4.10 (m, 1H), 3.96-3.67 (m, 3H), 3.47-3.2.99 (m, 2H), 2.75-2.64 (m,2H), 2.63-2.55 (m, 1H), 2.42-2.35 (m, 1H), 1.53-1.51 (m, 1H), 1.30-1.19(m, 1H), 1.10-1.07 (m, 1H), 0.83-0.76 (m, 6H). ¹³C NMR (DMSO-d₆, 100MHz) δ 172.7, 156.4, 56.3, 144.4, 144.3, 144.25, 144.17, 129.3, 128.7,128.6, 128.57, 128.00, 127.7, 127.5, 127.4, 126.2, 126.1, 125.6, 125.5,11.8, 120.5, 65.6, 52.7, 51.5, 48.2, 47.3, 41.6, 41.6, 34.7, 34.6, 34.1,31.3, 31.0, 24.7, 24.9, 23.8, 22.2, 21.9. HR-ESI: [M+H]⁺ cacl: 529.2697,found: 529.2695.3. Solid Phase Synthesis, Purification and Characterization ofγ-AApeptides

γ-AApeptides were prepared on Knorr resin in peptide synthesis vesselson a Burrell Wrist-Action shaker following standard Fmoc chemistryprotocol of solid phase peptide synthesis using synthesized γ-AApeptidebuilding blocks. Each coupling cycle included an Fmoc deprotection using20% Piperidine in DMF, and 8 h coupling of 1.5 equiv of γ-AApeptidebuilding blocks onto resin in the presence 4 equiv of DIC(diisopropylcarbodiimide)/DhbtOH(3-4-Dihydro-3-hydroxy-4-oxo-1-2-3-benzotriazine) in DMF. After desiredsequences were assembled, they were transferred into 4 ml vials andcleaved from solid support in 50:48:2 TFA/CH₂Cl₂/triisopropylsilaneovernight. Then solvent was evaporated and the residues were analyzedand purified on an analytical (1 ml/min) and a preparative (20 ml/min)Waters HPLC system, respectively, using 5% to 100% linear gradient ofsolvent B (0.1% TFA in acetonitrile) in A (0.1% TFA in water) over 40min, followed by 100% solvent B over 10 min. The HPLC traces weredetected at 215 nm. The desired fractions were eluted as single peaksat >95% purity. They were collected and lyophilized. The molecularweights of γ-AApeptides were obtained on a Bruker AutoFlex MALDI-TOFmass spectrometer using α-cyano-4-hydroxy-cinnamic acid (shown in Table2).

TABLE 2 MS analysis of γ-AApeptides (γ1-γ5). γ-AApeptides molecularweight (actual) molecular weight (found) γ1 927.2  927.4 (MALDI) γ21534.0 1535.0 (M + H)⁺ (LC-MS) γ3 2140.8 2141.3 (LC-MS) γ4 2125.8 2125.8(MALDI) γ5 2110.8 2111.7 (M + H)⁺ (MALDI)4. Antimicrobial Assays

The microbial organisms used were E. coli (JM109), B. subtilis (BR151),S. epidermidis (RP62A), C. albicans (ATCC 10231), E. faecalis (ATCC700802), S. aureus (ATCC 33592), K. pneumoniae (ATCC 13383),methicillin-resistant S. aureus (MRSA, USA100 lineage), and B.anthracis. The minimum inhibitory concentration (MIC) is the lowestconcentration that completely inhibits the growth of bacteria in 24 h.The highest concentration tested for antimicrobial activity was 100μg/ml. The antimicrobial activities of the γ-AApeptides were determinedin sterile 96-well plates by the broth micro-dilution method. Bacterialcells (Patch et al., 2003) and fungi (Karlsson et al., 2006) were grownovernight at 37° C. in 5 ml medium, after which a bacterial suspension(approximately 106 CFU/ml) or fungal suspension of Candida albicans(ATCC 10231) (approximately 103 CFU/ml) in Luria broth or trypticase soywas prepared. Aliquots of 50 μL bacterial or fungal suspension wereadded to 50 μL of medium containing the γ-AApeptides for a total volumeof 100 μL in each well. The γ-AApeptides were prepared in PBS buffer in2-fold serial dilutions, with the final concentration range of 0.5 to100 μg/ml. Plates were then incubated at 37° C. for 24 h (for bacteria)or 48 h (for Candida albicans (ATCC 10231)). The lowest concentration atwhich complete inhibition of bacterial growth (determined by a lack ofturbidity) is observed throughout the incubation time is defined as theminimum inhibitory concentration (MIC). The experiments were carried outindependently three times in duplicate.

5. Drug Resistance Study (Choi et al., 2009)

The initial MIC of γ5 and control antibiotic norfloxacin against S.aureus was obtained as described above. Bacteria from duplicate wells atthe concentration of one-half MIC were then used to prepare thebacterial dilution (approximately 10⁶ CFU/ml) for the next experiment.These bacterial suspensions were then incubated with γ5 and norfloxacinrespectively. After incubation at 37° C. for 24 h, the new MIC wasdetermined. The experiment was repeated each day for 17 passages.

6. Hemolysis Assay

Freshly drawn human red blood cells (hRBC's) with additive K₂ EDTA(spray-dried) were washed with PBS buffer several times and centrifugedat 1000 g for 10 min until a clear supernatant was observed. The hRBC'swere resuspended in 1×PBS to get a 5% v/v suspension. Two-fold serialdilutions of γ-AApeptides dissolved in 1×PBS from 250 μg/ml through 1.6μg/ml were added to a sterile 96-well plate to make up a total volume of50 μL in each well. Then 50 μL of 5% v/v hRBC solution was added to makeup a total volume of 100 μL in each well. The 0% hemolysis point and100% hemolysis point were determined in 1×PBS and 0.2% Triton-X-100,respectively (Patch et al., 2003). The plate was then incubated at 37°C. for 1 h and centrifuged at 3500 rpm for 10 min. The supernatant (30μL) was diluted with 100 μL of 1×PBS and absorption was detected bymeasuring the optical density at 360 nm by Biotek Synergy HT microtiterplate reader. % hemolysis was determined by the following equation:% hemolysis=(Abs_(sample)−Abs_(PBS))/(Abs_(Triton)−Abs_(PBS))×100H50 is the concentration of γ-AApeptide amphiphiles at which 50%hemolysis was observed. The highest concentration tested in thehemolytic assay was 500 μg/ml.7. Fluorescence Microscopy

A double staining method with DAPI (4′,6-Diamidino-2-phenylindoledihydrochloride, Sigma, >98%) and PI (Propidium iodide, Sigma) asfluorophores was used to visualize and differentiate the viable from thedead E. coli or B. subtilis cells. DAPI as a double-stranded DNA bindingdye stains all bacterial cells irrespective of their viability, whereasEthidium derivatives such as propidium iodide (PI) are capable ofpassing through only damaged cell membranes and intercalate with thenucleic acids of injured and dead cells to form a bright red fluorescentcomplex (Matsunaga et al., 2005). The cells were first stained with PIand then with DAPI. Bacterial cells were grown until they reached themid-logarithmic phase and then they (˜2×10⁶ cells) were incubated withthe γ-AApeptide γ5 at a concentration of 2×MIC (10 μg/ml) for 2 h. Thenthe cells were pelleted by centrifugation at 3000 g for 15 min in anEppendorf microcentrifuge. The supernatant was then decanted and thecells were washed with 1×PBS several times and then incubated with PI (5μg/ml) in the dark for 15 min at 0° C. The excess PI was removed bywashing the cells with 1×PBS several times. Then the cells wereincubated with DAPI (10 μg/ml in water) for 15 mins in the dark at 0° C.The DAPI solution was removed and cells were washed with 1×PBS severaltimes. Controls were performed following the exact same procedure forbacteria without the addition of γ5. The bacterial cells were thenexamined by using the Zeiss Axio Imager Z1 optical microscope with anoil-immersion objective (100×) (Williams et al., 1998).

EXAMPLE 2 Derivatization of γ-AApeptides

Lipidated and cyclic γ-AApeptides are also show potent andbroad-spectrum activity. The most potent linear sequence, γAA1 (γAA5 ofExample 1), is also listed in Table 3. For comparison, pexiganan (inPhase III clinical trials, a synthetic antimicrobial peptide) is alsoincluded; the data is taken from Chongsiriwatana et al. (2008),Chongsiriwatana et al. (2011), Ge et al. (1999) and Hicks et al. (2007).The results show that γAA1 (γAA5 of Example 1) and the lipidated andcyclic γ-AA peptides are generally comparable or more effective asantimicrobial agents. Lipidated γAA2 did not cause resistance to thedrug (lipidated γAA2; see FIG. 4B).

Structure:

TABLE 3 MIC (μg/ml) Organism γAA1 γAA2 γAA3 γAA4 γAA5 γAA6 γAA7Pexiganan Gram-negative E. coli 2.5-5   2.5 1.6-3.1 3.1-6.3 12.5-2525-50 2.5 16-32 K. pneumoniae 5 5 12.5-25   >100 8 10 50  8-16 (causingpneumonia) P. aeruginosa 25-50 25-50 3-6  6-12 8 10 2.5  6-12Gram-Positive B. subtilis 2 2.5 1.6-3.1 2 1 2 2 3.9 S. epidermidis(multi- 3.1-6.3 4 1.6-3.1 2 2 2 1  8-16 drug resistant) E. faecalis3.1-6.3 5 1.6-3.1 12.5-25   5 5 2.5 16-32 (vancomycin-resistant) S.aureus (methicillin- 5 4 1.6-3.1 3.1-6.3 1 3 5 16-32 resistant) MRSAUSA100 5 10 — — — — — — (leading hospital- associated strain,unresponsive to almost all antibiotics) B. anthracis (lethal 5 5 — — — —— — bioweapon) Fungi C. albicans  5-10 5 1.6-3.1 3-6 2 4 1.5 124Hemolysis (H₅₀) 300 >500 150 >500 100 300 >500 >500

EXAMPLE 3 The Design and Synthesis of Cyclic γ-AApeptides

The design of the cyclic antimicrobial γ-AApeptides of the currentinvention is based on the linear antimicrobial γ-AApeptides (FIG. 7)(Niu, Padhee et al., 2011; Padhee et al., 2011). Potent antimicrobialactivity can be achieved by joining amphiphilic building blocks togetherto form a globally amphipathic conformation upon interaction withbacterial membranes. The activity and selectivity can be fine-tuned byvarying the ratio of cationic/hydrophobic groups.

1. Synthesis and Characterization of γ-AApeptides Building Blocks

To achieve a global distribution of cationic and hydrophobic groupsalong the backbone, we prepared amphiphilic building blocks with acationic group and a hydrophobic group on either side (FIG. 7A). Byjoining these building blocks together and cyclizing the resultingoligomer (FIG. 7B), a global amphiphilicity is achieved upon binding tobacterial membranes (Niu, Padhee et al., 2011). The amphiphilic buildingblock 2 was prepared according to the previously published procedure(Niu, Padhee et al., 2011), in which the amino acid is lysine, and thephenyl-ended side chain is appended to the amine. The structures ofbuilding blocks 1, 2, 3, and 4 are shown below.

The γ-AApeptide building blocks (shown above) were synthesized followingthe previously reported procedure (Niu, Hu et al., 2011; Niu, Jones etal. 2011; Niu, Padhee et al., 2011). The characterization of buildingblock 2 has been reported (Niu, Padhee et al., 2011). The synthesis ofbuilding block 1 and 3 is also shown above. Given that an introductionof a hydrophobic building block can tune the overall amphiphilicity ofγ-AApeptides and improve their antimicrobial activity (Niu, Padhee etal., 2011), we also prepared building block 3 based on the reportedprocedure. To facilitate the on-resin cyclization of γ-AApeptide, aspecial γ-AApeptide building block 1 was designed. While the synthesiswas carried out similarly to the previously reported procedure (Niu,Padhee et al., 2011), the mono-allyl succinate was employed to modifythe amine.

Block 1. Yield 60% (two steps from 4). ¹H NMR (DMSO-d6, 400 MHz) δ (tworotamers) 7.88 (d, 2H), 7.62-7.57 (m, 2H), 7.42-7.29 (m, 4H), 7.28-7.15(m, 5H), 5.93-5.83 (m, 1H), 5.31-5.25 (m, 1H), 5.19-5.15 (m, 1H),4.52-4.49 (m, 2H), 4.21-4.03 (m, 4H), 3.88 (d, 2H), 3.63-3.35 (m, 2H),3.10-2.47 (m, 6H). ¹³C NMR (DMSO-d6, 100 MHz) δ 171.8, 171.7, 171.2,171.0, 170.6, 155.6, 155.6, 143.8, 143.7, 143.7, 143.7, 140.6, 140.6,138.7, 138.6, 132.6, 132.6, 129.0, 128.0, 127.9, 127.5, 126.9, 126.0,125.8, 125.0, 125.0, 120.0, 117.4, 117.4, 65.3, 64.2, 64.2, 51.7, 51.4,46.6, 46.5, 37.3, 28.9, 28.8, 27.4, 27.1. HR-ESI: [M+H]⁺ cacl: 571.2439,found: 571.2410.

Block 3. Yield 60%. ¹H NMR (DMSO-d6, 400 MHz) δ (two rotamers) 7.83 (d,J=7.6 Hz, 2H), 7.58-7.53 (m, 2H), 7.36-7.26 (m, 5H), 7.24-7.09 (m, 5H),4.13-4.06 (m, 3H), 3.96-3.74 (m, 4H), 3.51-3.46 (m, 1H), 3.40-3.32 (m,1H), 3.38-3.11 (m, 1H), 2.78-2.72 (m, 1H), 2.67-2.56 (m, 1H), 2.32-2.06(m, 2H), 1.47-1.36 (m, 1H), 1.33-1.24 (m, 2H), 0.80-0.71 (m, 6H). ¹³CNMR (DMSO-d6, 100 MHz) δ 173.7, 156.1, 144.2, 141.1, 139.5, 139.1,129.53, 129.46, 128.4, 128.0, 127.4, 126.5, 126.3, 125.7, 125.6, 125.5,120.5, 120.5, 65.8, 65.8, 51.5, 51.0, 47.0, 38.3, 34.3, 34.1, 30.9,30.9, 27.6, 27.5, 22.8, 22.7. HR-ESI: [M+H]⁺ cacl: 529.2697, found:529.2700.

Block 4. Yield 82%. ¹H NMR (CDCl3, 400 MHz) δ 7.72 (d, J=8 Hz, 2H),7.49-7.45 (m, 2H), 7.38-7.34 (m, 2H), 7.26-7.14 (m, 7H), 6.18-6.14 (m,1H), 4.26-4.16 (m, 3H), 4.09-4.06 (m, 1H), 3.78-3.68 (m, 2H), 3.42-3.37(m, 1H), 3.17-3.15 (m, 1H), 2.99-2.94 (m, 1H), 2.86-2.81 (m, 1H), 1.39(s, 9H). ¹³C NMR (CDCl3, 100 MHz) δ 165.2, 165.1, 162.1, 161.7, 156.9,156.8, 143.9, 143.6, 141.2, 141.1, 136.0, 129.0, 128.8, 127.7, 127.6,127.1, 125.2, 119.8, 84.8, 67.3, 50.6, 49.8, 47.8, 46.8, 38.6, 36.9,36.8, 27.8, 27.7. HR-ESI: [M+H]⁺ cacl: 487.2591, found: 487.2565.

2. Solid Phase Synthesis, Purification and Characterization of Cyclicγ-AApeptides

Cyclic γ-AApeptides were prepared on a Rink amide resin in peptidesynthesis vessels, on a Burrell Wrist-Action shaker, following thestandard Fmoc chemistry protocol of solid phase peptide synthesis.Synthesized γ-AApeptide building blocks were used (FIG. 6A). Eachcoupling cycle included an Fmoc deprotection using 20% piperidine inDMF, and 8 h coupling of 1.5 equiv of γ-AApeptide building blocks in thepresence of 4 equiv of DIC (diisopropylcarbodiimide)/DhbtOH(3-4-Dihydro-3-hydroxy-4-oxo-1-2-3-benzotriazine) in DMF. Thecyclization was achieved on resin via the γ-AApeptide building block 1.Block 1 was first attached to the solid support, followed by standardFmoc solid phase synthesis. After desired sequences were assembled, theallyl group was removed by treatment of Pd(PPh₃)₄ (0.2 equiv)/PhSiH₃ (10equiv)/CH₂Cl₂ for 2 h (repeated two times). The deprotection of the Fmocgroup was then carried out on the N-terminus. The intramolecularcyclization was accomplished using PyBop/HOBt/DIEA/DMF. Next, the resinwas transferred into 4 mL vials and cyclic γ-AApeptides were cleavedfrom solid support in 50:48:2 TFA/CH2Cl2/triisopropylsilane overnight.Then solvent was evaporated and the residues were analyzed and purifiedon an analytical (1 mL/min) and a preparative (20 ml/min) Waters HPLCsystem, respectively, using 5% to 100% linear gradient of solvent B(0.1% TFA in acetonitrile) in A (0.1% TFA in water) over 40 min,followed by 100% solvent B over 10 min. The HPLC traces were detected at215 nm. The desired fractions were eluted as single peaks at >95% puritywith yields of 6-10% (based on loading of the resin, see FIG. 6B forsequences). They were collected and lyophilized. The molecular weightsof cyclic γ-AApeptides (Table 4) were obtained on a Bruker AutoFlexMALDI-TOF mass spectrometer using α-cyano-4-hydroxy-cinnamic acid.

TABLE 4 MALDI analysis of cyclic γ-AApeptides Cyclic γ- Yield (based onMolecular Molecular weight AApeptides loading of the resin) weight(actual) (found) HW-B-3  10.5% 1501.2 1053.1 (M + H⁺) HW-B-4  8.6%1805.1 1806.6 (M + H⁺) HW-B-5  6.2% 2108.3 2109.6 (M + H⁺) HW-B-11 6.8%2093.3 2147.6 (M + 3NH4⁺) HW-B-12 6.5% 2078.3 2079.9 (M + H⁺) HW-B-136.0% 2078.3 2079.0 (M + H⁺) HW-B-14 6.4% 2063.3 2117.3 (M + 3NH4⁺)3. Antimicrobial Assays

The microbial organisms used were B. subtilis (BR151), multi-drugresistant S. epidermidis (RP62A), C. albicans (ATCC 10231),vancomycin-resistant E. faecalis (ATCC 700802), methicillin-resistant S.aureus (ATCC 33592), and K. pneumoniae (ATCC 13383), multi-drugresistant P. aeruginosa ATCC 27853. The minimum inhibitory concentration(MIC) is the lowest concentration that completely inhibits the growth ofbacteria in 24 h. The highest concentration tested for antimicrobialactivity was 50 μg/mL. The antimicrobial activities of the cyclicγ-AApeptides were determined in a sterile 96-well plates by the brothmicro-dilution method. Bacterial cells 4 and fungi 5 were grownovernight at 37° C. in 5 mL medium, after which a bacterial suspension(approximately 106 CFU/mL) or fungal suspension of Candida albicans(ATCC 10231) (approximately 103 CFU/mL) in Luria broth or trypticase soywas prepared. Aliquots of 50 μL bacterial or fungal suspension wereadded to 50 μL of medium containing the cyclic γ-AApeptides for a totalvolume of 100 μL in each well. The cyclic γ-AApeptides were prepared inPBS buffer in 2-fold serial dilutions, with the final concentrationrange of 0.5 to 50 μg/mL. Plates were then incubated at 37° C. for 24 h(for bacteria) or 48 h (for Candida albicans (ATCC 10231)). The lowestconcentration at which complete inhibition of bacterial growth(determined by a lack of turbidity) is observed throughout theincubation time is defined as the minimum inhibitory concentration(MIC). The experiments were carried out independently three times induplicate.

Cyclized γ-AApeptides (HW-B-3, -4, -5) comprising four, five, and sixamphiphilic building blocks were prepared as an initial attempt, andtested for their antimicrobial activities against a series ofGram-positive and Gram-negative bacteria and fungi, many of which aremulti-drug resistant and clinically relevant strains (Table 5). Theoligomers' hemolytic activities toward human red blood cells were alsomeasured, as an indication of their selectivity. For comparison,Pexiganan, a phase III antimicrobial peptide drug candidate(Chongsiriwatana et al., 2008; Chongsiriwatana et al., 2011; Ge et al.,1999; Hicks et al., 2007), as well as γ5, the most potent linearγ-AApeptide (Niu, Padhee et al., 2011), were both used as controls.Similar to a linear γ-AApeptide, which appeared to be more potent with alonger sequence (Niu, Padhee et al., 2011), the cyclic γ-AApeptide withan increasing ring size (from HW-B-3 to HW-B-5) tended to augment theantimicrobial activities (Table 5). The most potent cyclic γ-AApeptide,HW-B-5, has a similar activity to the well-known Pexiganan, though it isstill inferior to γ5. It is notable that the hemolytic activity ofHW-B-5 is much less than Pexiganan and γ5, implying the potential toimprove its anti-bacterial activities through the introduction ofhydrophobic building blocks (Niu, Padhee et al., 2011).

Given that linear γ5 bears hydrophobic building blocks, and thusexhibited much more potent antimicrobial activities than other linearγ-AApeptides completely made from amphiphilic building blocks (Niu,Padhee et al., 2011), similar configurations for cyclic γ-AApeptideswere attempted. HW-B-11, -12, and -13 were thereby prepared toincorporate the same number of building blocks as HW-B-5, but have oneor two amphiphilic building blocks replaced by hydrophobic ones (FIG.6B). As a result, HW-B-11, with the change of only one building block,showed enhanced antimicrobial activities, especially againstGram-positive bacteria, which are comparable to linear γ5, and betterthan Pexiganan (Table 5). In spite of its weaker activity towardsGram-negative strain K. pneumoniae, HW-B-11 has a stronger inhibition offungus C. albicans than both Pexiganan and linear γ5, with a significantminimum inhibitory concentration (MIC) value of 5 mg/ml. In addition,HW-B-11 still possessed a low hemolytic activity, thereby making it apromising antimicrobial agent with comparable or even better selectivitythan Pexiganan and linear γ5. Further, HW-B-12 was developed toincorporate two hydrophobic building blocks, which are separated by twohydrophilic building blocks, in a way similar to the design of linearγ5. Its antimicrobial activities were not improved, or maybe weakercompared to HW-B-11. To assess whether the decrease in activity is dueto the incorporation of more hydrophobic building blocks or is caused bytheir relative positions in the ring, HW-B-13 was synthesized by placingtwo hydrophobic building blocks adjacent to each other (FIG. 6B).HW-B-13 exhibited even better activity than HW-B-11, Pexiganan, andlinear γ5 to arrest the growth of both Gram-positive and Gram-negativebacteria pathogens, as well as in fungus (Table 5). HWB-13 is a potentantimicrobial peptidomimetic with broad-spectrum activity, especiallytoward two of the most clinically relevant strains, S. aureus (MRSA) andP. aeruginosa (PA), with a MIC value of 1 mg/mL and 8 mg/mL,respectively: which is at least 5-fold more potent than the linear γ5.Though HW-B-13 appears to be more hemolytic, its overall selectivity inseveral important pathogens is still improved relative to linear γ5 andPexiganan.

To further investigate the effect of hydrophobicity and to tune theactivity, HW-B-14 (FIG. 6B) with three hydrophobic building blocks wasdeveloped, which, however, resulted in a slightly decreasedantimicrobial activity and hemolytic activity (Table 5) in comparison toHW-B-13. Nevertheless, the activity and selectivity of HW-B-14 are stillgenerally comparable, or superior to linear γ5, against several strainsincluding MRSA and PA.

TABLE 5 The antimicrobial and hemolytic activities of oligomers. TheMICs of Pexiganan and Linear γ5 (described above) are shown forcomparison. MIC (mg/ml) HW- HW- HW- HW- Linear Organism HW-B-3 BW-B-4HW-B-5 B-11 B-12 B-13^(a) B-14^(a) Pexiganan γ5 Gram-positive B.subtilis 25-50 10 5 2 5 1 2 4 2 S. epidermidis >50 10 8 2 5 2 2 8 5(MRSE) E. faecalis >50 20 20 15 8 5 5 32 5 (VREF) S. aureus >50 >5025-50 5 6 1 3 16 5 (MRSA) Gram-negative K. pneumoniae >50 >50 >50 20 5 810 8 5 P. aeruginosa >50 20 18 10 >50 8 10 8-16 >50 Fungus C.albicans >50 >50 >50 5 5 2 4 124 8Hemolysis >500/>500 >500/>500 >500/>500 200/>500 150/450 40/100 45/300181/495 75/300 (H10/H50) ^(a)Sequences showing the most broad-spectrumantimicrobial activity.

Based on these results, it appears that the inclusion of two neighboringhydrophobic building blocks brings in the optimal antimicrobialactivity. The structure-activity studies of HW-B-5, -11, -13, and -14suggest that a higher percentage of hydrophobic groups in γ-AApeptidesleads to greater antimicrobial activity. It is well accepted that morelipophilicity would lead to increased hemolytic activity(Chongsiriwatana et al., 2008; Mowery et al., 2009). While the result ofour structure-activity studies generally supports this rule, theslightly decreased hemolytic activity demonstrated by HW-B-14, which hasone more hydrophobic building block than HW-B-13, is quite unexpected.It suggests that besides the absolute hydrophobicity of functionalgroups, the overall conformations of molecules may also affect theirhemolytic activity.

In this case, multiple neighboring hydrophobic building blocks inHW-B-14 promote hydrophobic interactions among their side chains, whichlimit their efficient contact with red blood cells. Finally, thedistinct activities between HW-B-12 and HW-B-13 suggest the importanceof position for hydrophobic building blocks. A preliminary computermodeling of HW-B-13 reveals that the cyclic γ-AApeptide naturally adoptsa globally amphipathic conformation, with cationic side groups clusteredat the bottom left face of the ring, and the majority of hydrophobicgroups at the top face of the ring (FIG. 8). Such a constrainedstructure with predefined amphiphilicity may favor the binding anddisruption events within bacteria membranes. To the contrary, theamphiphilic topology of HW-B-12 may be scrambled by the separatedhydrophobic building blocks. Though linear γ-AApeptides with scrambledamphipathicity can still be induced by a membrane to adopt a globalamphiphilicity (Niu, Padhee et al., 2011), the rigid structure of cyclicγ-AApeptides compromises their conformational flexibility.

4. MTT Cytotoxicity Assay

N2a APP cells were used to access the cytotoxicity of cyclicγ-AApeptides toward mammalian cells (FIG. 9). Typically, stockconcentration of a particular cyclic γ-AApeptide (1 mg/ml) was dilutedin media to make different concentrations in 96-well plates, and thenincubated at 37° C. Following that, N2a APP cells were seeded to 1×10⁴cells/well in 100 μl media in another 96-well plate. After incubationfor 12 hours, 100 μl of different concentrations of cyclic γ-AApeptideswere added and the plate was incubated for another 36 hours. The mediain the 96-well plate was removed and washed with fresh media once, and110 μl MTT reagent was added. The mixture was incubated for another 4hours, after which 100 μl of pre-warmed solubilization solution wasadded. The plate was then incubated at 37° C. for 12 h, and theabsorbance at 550 nm was read. Percentage of cell viability wascalculated based on the following equation:cell viability %=(A/A _(control))×100

The results are quite consistent with hemolytic results, with morehemolytic sequences being more toxic. However, the EC₅₀s (the effectiveconcentration to cause half of the cells' death) are all above 12.5mg/mL, indicating cyclic γ-AApeptides are very selective toward bacteriaover mammalian cells. For instance, HW-B-13 is at least 12.5-fold moreselective toward MRSA.

5. Lipid Depolarization

To understand the antimicrobial mechanisms of cyclic γ-AApeptides, themost active ones, HW-B-11, -13, and -14, were used to investigate theireffects in cytoplasmic membrane disruption through the depolarization ofthe S. aureus membrane (FIG. 10) (Choi et al, 2009). The lipiddepolarization of the bacterial cell membrane was conducted using themembrane potential sensitive dye 3, 5′-dipropylthiacarbocyanine iodide(DiSC3-5) that distributes between the cells and the medium depending onthe membrane potential gradient. S. aureus (ATCC 33592) cells were grownin Luria broth and Trypticase soy broth medium respectively to amid-logarithmic phase (OD600=0.5-0.6). The bacterial cells were thencollected by centrifugation at 3000 rpm for 10 min and then washed oncewith buffer (5 mM HEPES and 5 mM glucose, pH 7.2). The cells werere-suspended to OD600=0.05 with 100 mM KCl, 2 μM DiSC3-5.5 mM HEPES and5 mM glucose and were incubated for 30 min at 37° C. for maximal dyeuptake and fluorescence self-quenching. This bacterial suspension (90μL) and 10 μL of compound stock solutions or control drug solution wereadded to white flat-bottomed polypropylene 96-well plates (Costar) andincubated at 37° C. for 30 min. The fluorescence reading was monitoredusing the microplate reader (Biotek) at an excitation wavelength of 622nm and an emission wavelength of 670 nm); the fluorescence increased dueto the disruption of cytoplasmic membrane. Valinomycin (finalconcentration 250 μg/mL) was used as a positive control, and the blankwith only cells and dye was used as the background.

The distribution of DiSC3-5 between the medium and the cell interiorreflects the membrane potential (Choi et al, 2009). The loss of membranepotential as a result of membrane permeation/disruption leads to adramatic increase in fluorescent intensity (Choi et al, 2009). Althoughthe oligomer concentration needed for depolarization is actually higherthan the oligomers' MIC values, generally more active antimicrobialoligomers with lower MIC values tended to reach a high percentage ofdepolarization at a lower concentration. Such a trend was clearlydemonstrated by HW-B-11, -13 and -14, which supports the membranedisruption mechanism of cyclic γ-AApeptides (FIG. 10).

6. Fluorescence Microscopy

The antimicrobial mechanism of cyclic γ-AApeptides was further assessedby fluorescence microscopy, in which B. subtilis was treated with themost potent HW-B-13, and stained with DAPI and PI33 dyes (FIG. 11). Thedouble staining method with DAPI (4′, 6-Diamidino-2-phenylindoledihydrochloride, Sigma, >98%) and PI (Propidium iodide, Sigma) asfluorophores was used to visualize and differentiate the viable from thedead B. subtilis cells. DAPI, as a double-stranded DNA binding dye,stains all bacterial cells irrespective of their viability, whereasEthidium derivatives such as propidium iodide (PI) are capable ofpassing through only damaged cell membranes and intercalating with thenucleic acids of injured and dead cells to form a bright red fluorescentcomplex (Matsunaga et al., 1995). The cells were first stained with PIand then with DAPI. Bacterial cells were grown until they reached themid-logarithmic phase and then they (˜2×10⁶ cells) were incubated withthe cyclic γ-AApeptide HW-B-13 at the concentration of 2×MIC (10 μg/mL)for 2 h. Then the cells were pelleted by centrifugation at 3000 g for 15min in an Eppendorf microcentrifuge. The supernatant was then decantedand the cells were washed with 1×PBS several times and then incubatedwith PI (5 μg/mL) in the dark for 15 min at 0° C. The excess PI wasremoved by washing the cells with 1×PBS several times. Then the cellswere incubated with DAPI (10 μg/mL in water) for 15 min in the dark at0° C. The DAPI solution was removed and the cells were washed with 1×PBSseveral times. Controls without the addition of HW-B-13 were performedfollowing exactly the same procedure for bacteria. The bacterial cellswere then examined by using the Zeiss Axio Imager Z1 optical microscopewith an oil-immersion objective (100×) (Niu, Padhee et al., 2011; Padheeet al., 2011; Williams et al., 1998).

Compared to little PI staining (red fluorescence) observed in thecontrol group, B. subtilis incubated with HW-B-13 for 2 h displayed astrong red staining by PI, indicating the significant disruption ofbacterial membranes by HW-B-13. The aggregation of dead bacterial cellsafter the oligomer treatment is consistent with literature reports,(Niu, Padhee et al., 2011; Chen et al., 2010), an indication of loss ofmembrane potential.

EXAMPLE 4 The Design and Synthesis of Lipidated-γ-AApeptides

1. Synthesis of the γ-AApeptide Building Blocks for Lipidatedγ-AApeptides

The γ-AApeptide building blocks used in the solid phase synthesis oflipidated γ-AApeptides were synthesized following the previouslyreported procedure (Niu, Hu et al., 2011; Niu, Jones et al., 2011; Niu,Padhee et al., 2011). The examples of the building blocks are shownbelow.

Compound B1. Yield 65.3% in two steps from the secondary amine. 1-3 ¹HNMR (400 MHz, DMSO-d6) δ 7.84 (d, J=8.0 Hz, 2H), 7.64-7.61 (dd, J=4.0,8.0 Hz, 2H), 7.37 (t, J=8.0 Hz, 2H), 7.28 (t, J=8.0 Hz, 2H), 7.14 & 6.98(2d, J=8.0 Hz, 1H), 6.74-6.69 (m, 1H), 4.29-4.14 (m, 3H), 3.40-3.81 (m,2H), 3.62-3.52 (m, 2H), 3.28-3.16 (m, 1H), 2.95-2.69 (m, 4H), 2.36-2.22(m, 1H), 2.12-2.01 (m, 1H), 1.70-1.13 (m, 26H). ¹³C NMR (100 MHz, CD3OD)δ 174.7, 174.4, 171.5, 171.2, 157.32, 157.3, 157.1, 143.9, 143.8,141.23, 141.20, 127.3, 126.7, 124.7, 124.6, 119.5 78.5, 78.4, 66.1,65.9, 53.1, 51.2, 50.1, 49.9, 49.8, 39.8, 39.5, 39.3, 31.6, 31.2, 29.7,29.5, 29.2, 27.4, 25.2, 22.9, 22.8. HR-ESI: [M+H]⁺ cacl: 697.3807,found: 697.3796.

Compound B2. Yield 82.6% in two steps from the secondary amine. 1-3 ¹HNMR (400 MHz, DMSO-d6) δ 7.83 (d, J=8.0 Hz, 2H), 7.62 (d, J=7.6 Hz, 2H),4.28-4.14 (m, 3H), 4.03-3.63 (m, 2H), 3.42-3.33 (m, 2H), 3.22-3.21 (m,1H), 1.98 & 1.84 (rotamers, 2s, 3H), 1.02 & 0.95 (rotamers, 2d, J=6.4Hz, 3H). ¹³C NMR (100 MHz, DMSO-d6) δ 171.6, 171.4, 171.2, 170.7, 156.1,144.3, 144.26, 141.2, 128.0, 127.5, 125.6, 125.5, 120.5, 65.7, 65.6,56.4, 54.2, 51.5, 51.1, 49.97, 47.2, 17.17, 45.9, 21.7, 21.4, 18.8, 18.5HR-ESI: [M+H]⁺ cacl: 397.1758, found: 397.1760.

Compound B3. Yield 75.9% in two steps from the secondary amine. 1-3 ¹HNMR (400 MHz, DMSO-d6) δ 7.86-7.83 (m, 2H) 7.65-7.60 (m, 2H), 7.39-7.13(m, 2H), 7.14 & 7.00 (d&d, J=8.0 Hz, 1H), 6.75-6.72 (m, 1H), 4.31-4.22(m, 2H), 4.17-4.12 (m, 1H), 3.97-3.77 (m, 2H), 3.72-3.64 (m, 1H),3.46-3.18 (m, 2H), 2.91-2.84 (m, 2H), 2.39-2.24 (m, 1H), 2.16-2.02 (m,1H), 1.57-1.50 (m, 2H), 1.38-1.26 (m, 11H), 0.86-0.77 (m, 6H). ¹³C NMR(100 MHz, DMSO-d6) δ 173.0, 172.7, 171.2, 156.4, 156.3, 156.0, 144.4,144.35, 144.3, 144.2, 141.2, 141.1, 128.0, 127.44, 127.41, 125.5, 120.5,79.6, 77.7, 65.6, 65.4, 52.9, 51.3, 50.7, 48.3, 47.32, 47.26, 41.6,41.0, 30.2, 29.8, 25.7, 25.6, 24.7, 23.8, 23.7, 22.1, 21.9. HR-ESI:[M+H]⁺ cacl: 582.3174, found: 582.3175.

Compound B4. Yield 63.8% in two steps from the secondary amine. 1-3 ¹HNMR (400 MHz, DMSO-d6) δ 7.86-7.80 (m, 4H), 7.40-7.29 (m, 4H), 4.02-3.93(m, 2H), 3.73-3.69 (m, 1H), 3.68-3.62 (m, 1H), 3.53-3.50 (m, 1H),3.49-3.36 (m, 1H), 3.19-3.14 (m, 1H), 3.13-3.02 (m, 1H), 2.35-2.22 (m,2H), 1.72-1.60 (m, 2H), 1.37 & 1.36 & 1.35 (rotamers, m, 18H). ¹³C NMR(100 MHz, DMSO-d6) δ 172.4, 167.8, 167.3, 156.4, 156.3, 144.4, 144.2,141.1, 128.0, 127.5, 127.47, 125.63, 125.61, 120.5, 80.7, 79.8, 65.7,60.2, 53.0, 50.6, 50.4, 49.9, 49.4, 49.1, 47.2, 42.7, 42.4, 42.1, 42.1,42.0, 31.9, 31.3, 28.2, 28.1, 28.0 HR-ESI: [M+H]⁺ cacl: 611.2963, found:611.2958.

2. Solid Phase Synthesis, Purification and Characterization of Lipidatedγ-AApeptides (Niu, Hu et al., 2011; Niu, Jones et al., 2011; Niu, Padheeet al., 2011)

The lipidated γ-AApeptides (also referred to as lipo-γ-AApeptides) wereprepared by attaching hydrophobic alkyl tails to cationic γ-AApeptides.The synthesis of lipidated γ-AApeptides was carried out on the solidphase (as shown below) and purified by HPLC adapted from previouslyreported protocols (Niu, Padhee et al., 2011; Niu, Jones et al., 2011;Niu, Hu et al., 2011).

Lipidated γ-AApeptides were prepared on a Rink amide resin in peptidesynthesis vessels on a Burrell Wrist-Action shaker following thestandard Fmoc chemistry of solid phase peptide synthesis protocol. Eachcoupling cycle included an Fmoc deprotection using 20% piperidine inDMF, and 4 h coupling of 1.5 equiv of γ-AApeptide building blocks (Niu,Hu et al., 2011; Niu, Jones et al., 2011; Niu, Padhee et al., 2011) ontothe resin in the presence of 2 equiv of DIC(diisopropylcarbodiimide)/DhBtOH (oxohydroxybenzotriazole) in DMF. Thelipidation was accomplished by reacting lauric acid, palmitic acid oroleic acid with N-terminus using DIC/DhBtOH as activation agents on thesolid phase. After the desired sequences were assembled, they weretransferred into a 4 ml vial and cleaved from solid support in 50:45:5TFA/CH₂Cl₂/triisopropylsilane overnight (FIG. 12). Then the solvent wasevaporated and the residues were analyzed and purified on a Waters HPLCinstalled with both an analytical module (1 ml/min) and a preparativemodule (20 ml/min). Both modules had the same methods which were using5% to 100% linear gradient of solvent B (0.1% TFA in acetonitrile) in A(0.1% TFA in water) over 40 min, followed by 100% solvent B over 10 min.The desired fractions were generally over 70% in crude (determined byHPLC) and eluted as single peaks at >95% purity. They were collected andlyophilized. The molecular weights of lipidated γ-AApeptides wereobtained on a Bruker AutoFlex MALDI-TOF mass spectrometer usingα-cyano-4-hydroxy-cinnamic acid as the matrix (shown in Table 6).

TABLE 6 MALDI analysis of lipidated γ-AApeptides Lipo-γ- AApeptideFormula Mass calculated Mass found 1 C₂₄H₄₉N₅O₃ 455.38 458.20 (M + 3H)⁺2 C₂₈H₅₇N₅O₃ 511.4  512.6 (M + H)⁺ 3 C₄₀H₈₁N₉O₅ 767.6  768.7 (M + H)⁺ 4C₅₂H₁₀₅N₁₃O₇ 1023.8 1047.0 (M + Na)⁺ 5 C₆₆H₁₂₉N₁₇O₁₁ 1336.0 1336.9 (M +H)⁺ 6 C₆₆H₁₂₆N₁₄O₁₁ 1291.0 1293.7 (M + H)⁺ 7 C₆₀H₉₉N₁₁O₂₃ 1341.6 1358.7(M + NH4)⁺ 8 C₇₂H₁₀₅N₁₁O₁₇ 1395.8 1418.3 (M + Na)⁺ 9 C₅₀H₉₆N₁₄O₁₀ 1053.41054.0 (M + H)⁺ 10 C₃₀H₅₉N₅O₃ 537.5  538.6 (M + H)⁺ 11 C₃₆H₇₁N₇O₄ 666.0 666.6 (M + H)⁺ 12 C₄₂H₈₃N₉O₅ 793.7  816.8 (M + Na)⁺ 13 C₆₈H₁₂₈N₁₄O₁₁1317.0 1318.5 (M + H)⁺

Examples of lipidated γ-AApeptides of the current invention aredescribed in FIG. 12, named as sequence 1 to sequence 13. Sequences 1-6are cationic lipidated γ-AApeptides with saturated lauric acid orpalmitic acid tails. Sequences 7 and 8 are anionic lipidatedγ-AApeptides that were prepared as negative controls. Sequence 9contains the same γ-AApeptide sequence as 6, yet lacks an alkyl tail.Sequences 10-13 are cationic γ-AApeptides alkylated with unsaturatedoleic acid.

3. Antimicrobial Assays

The sequences shown in FIG. 12 were tested for their antimicrobialactivity toward a range of Gram-positive and Gram-negative bacteria, aswell as the fungus C. albicans. Their selectivity was also evaluated viahemolytic assays. The results of these investigations are shown in Table7, with γ5, a linear γ-AApeptide reported previously (Niu, Padhee etal., 2011), included for comparison.

The bacterial strains used in the experiment were E. coli (JM109), B.subtilis (BR151), multi-drug resistant S. epidermidis (RP62A),vancomycin-resistant E. faecalis (ATCC 700802), methicillin-resistant S.aureus (ATCC 33592), K. pneumoniae (ATCC 13383), and multi-drugresistant P. aeruginosa ATCC 27853. The fungal strain used was C.albicans (ATCC 10231). The antimicrobial activities of the lipidatedγ-AApeptides were determined in a sterile 96-well plates by the brothmicro-dilution method. Bacterial cells (Patch et al., 2003) and fungi(Karlsson et al., 2006) were grown overnight at 37° C. in 5 ml medium,after which a bacterial suspension (approximately 106 CFU/ml) or fungalsuspension of Candida albicans (ATCC 10231) (approximately 103 CFU/ml)in Luria broth or trypticase soy was prepared. Aliquots of 50 μLbacterial or fungal suspension were added to 50 μL of medium containingthe lipidated γ-AApeptides for a total volume of 100 μL in each well.The lipidated γ-AApeptides were prepared in PBS buffer in 2-fold serialdilutions, with the final concentration range of 0.5 to 100 jag/ml.Plates were then incubated at 37° C. for 24 h (for bacteria) or 48 h(for Candida albicans (ATCC 10231). The lowest concentration at whichcomplete inhibition of bacterial growth (determined by a lack ofturbidity) is observed throughout the incubation time is defined as theminimum inhibitory concentration (MIC). The experiments were carried outindependently three times in duplicate.

As shown in Table 7, almost all the cationic lipidated γ-AApeptidesdisplay broad-spectrum antimicrobial activity against an array ofGram-negative and Gram-positive bacteria, as well as toward the fungusC. albicans. Sequences 7, 8, and 9, however, are not active at all underthese experimental conditions. This is likely because, with a positivelycharged surface post-self-assembly, cationic lipidated γ-AApeptidesselectively target bacteria that have negatively charged membranes.Sequences 7 and 8, which are negatively charged lipidated γ-AApeptides,should not, and do not, present any activity because of electrostaticrepulsion with bacterial membranes. Linear sequence 9, althoughpositively charged, cannot strongly interact with bacterial membranesbecause of its lack of a lipid tail.

TABLE 7 Antimicrobial Activities of γ-AApeptides^(a) MIC (μg/ml) Hemo-Gram negative Gram positive Fungi, lysis oligo- E. K. P. B. S. E. S. C.HC10/ selec- mers coli pneumoniae aeruginosa subtilis epidermidisfaecalis aureus albicans HC50 tivity^(b) 1 >50 >50 >50 >50 >50 >50 >50 >50 400/>500  2 2.5 35 10 1.5 2 2.5 2.5 225/40  16  3 30 20 20 1.5 2.5 8 5 2 40/300 60  4 20 >50 15 1.5 2 10 5 2300/>500 >100  5 2.5 15 15 2.5 10 10 10 7.5 200/>500 >50  6* 2.5 5 5 2.54 5 4 5  60/>500 >125  7 >100 >100 >100 >100 >100 >100 >100 >100250/>500  8 >100 >100 >100 >100 >100 >100 >100 >100 200/>500 9 >100 >100 >100 >100 >100 >100 >100 >100 120/>500 10 2.5 35 3 2 2 2.54 2 40/100 25 11 10 40 15 2 2 2.5 5 5  80/>500 >100 12 10 15 3 2.5 4 5 52 250/>500 >100  13* 3 3 3 3 3 4 3 3 100/>500 >167 γ5 3 5 >50 2 5 5 5 875/300 60 ^(a)HC10 and HC50 are the concentrations of γ-AApeptides atwhich 10% or 50% hemolysis was observed. The two most potent andbroad-spectrum lipo-γ-AApeptides, 6 and 13, are designated with an *.^(b)Selectivity is calculated based on H50/the MIC of S. aureus.

To investigate further development of lipidated γ-AApeptides basedantimicrobial agents, the structure-function relationship of thesecompounds was investigated. Sequence 1, bearing one cationic γ-AApeptidebuilding block and a lauric acid alkyl tail, is not active toward anymicroorganism. However, changing the lauric acid alkyl tail to apalmitic alkyl chain renders Sequence 2 a potent antimicrobial agenttoward most bacteria, as well as the fungus C. albicans. This indicatesthat lipophilicity of the alkyl tail is highly important for interactionwith cell membranes and that increased cationic charge does notnecessarily lead to more potent antimicrobial agents. Sequence 3 andSequence 4, which have many more cationic charges than Sequence 2, areseemingly less active toward bacteria and fungus. The balance ofhydrophobicity and cationic charge appears to affect the antimicrobialactivity. This led to the discovery of Sequence 5 and Sequence 6, withtwo more hydrophobic γ-AApeptide building blocks, which are more broadlyactive. Sequence 6, which is a lipidated version of Sequence 9,effectively arrests the growth of all tested bacteria and fungi.However, as lipophilicity increases, hemolytic activity also increases,compromising selectivity. To enhance selectivity, Sequences 10-13, whichcontain similar or identical sequences to Sequences 2-6 but withunsaturated oleic tails, were prepared. Unsaturated tails may havelesser propensity for aggregation but still possess the samehydrophobicity compared to their saturated counterparts.

A number of even more potent and broad-spectrum-active lipidatedγ-AApeptides were obtained, some of which are much less hemolytic, asseen with Sequence 10, Sequence 12, and Sequence 13. For example,Sequence 3 and Sequence 12 only differ in their alkyl tails, yetSequence 3 is somewhat hemolytic, while Sequence 12 is virtuallynonhemolytic, and is generally more active towards bacteria. Sequence13, being the most potent and broad-spectrum sequence, has similar andbetter antimicrobial activity than Sequence 6 toward all microorganisms,while it is again much less hemolytic. Such increased potency andenhanced selectivity may indicate that compared to membranes ofmammalian cells bacterial membranes are more sensitive to unsaturatedalkyl tails. This finding is significant for the development of novellipoantibiotics. Both Sequence 13 and Sequence 6 are more potent andbroad-spectrum than the linear sequence γ5 (Niu, Padhee et al., 2011),particularly toward the Gram-negative bacterium P. aeruginosa and fungusC. albicans, although they contain shorter lengths of γ-AApeptidefragments. Furthermore, Sequence 13 is less hemolytic than γ5. Theseresults indicate that bacteria and fungi are more sensitive andsusceptible to hydrophobic lipid chains, which supports the use oflipidated γ-AApeptides as novel antibiotic agents.

4. Hemolytic Assays

The sequences shown in FIG. 12 were also evaluated via hemolytic assays.Freshly drawn human red blood cells (hRBC's) with additive K2 EDTA(spray-dried) were washed with PBS buffer several times and centrifugedat 1000 g for 10 min until a clear supernatant was observed. The hRBC'swere resuspended in 1×PBS to get a 5% v/v suspension. Two-fold serialdilutions of lipidated γ-AApeptides dissolved in 1×PBS from 1 mg/mlthrough 6.3 μg/ml were added to sterile 96-well plates to make up atotal volume of 50 μL in each well. Then 50 μL of 5% v/v hRBC solutionwas added to make up a total volume of 100 μL in each well. The 0%hemolysis point and 100% hemolysis point were determined in 1×PBS and0.2% Triton-X-100, respectively (Patch et al., 2003). The plate was thenincubated at 37° C. for 1 h and centrifuged at 3500 rpm for 10 min. Thesupernatant (30 μL) was diluted with 100 μL of 1×PBS and absorption wasdetected by measuring the optical density at 360 nm with a BiotekSynergy HT microtiter plate reader. % hemolysis was determined by thefollowing equation:% hemolysis=(Abs sample−Abs PBS)/(Abs Triton−Abs PBS)×100

The results of the hemolytic assays are shown in Table 7.

5. Fluorescence Microscopy

To probe the antimicrobial mechanism of lipo-γ-AApeptide activity,fluorescence microscopy was performed to assess the ability of Sequence13 (FIG. 13) and Sequence 6 (FIG. 14) to cause membrane leakage, sincemembrane disruption is a general function of AMPs. The double stainingmethod with DAPI and PI was used, where DAPI stains all bacterial cellsirrespective of their viability and PI only stains injured or dead cellswith compromised membranes (Chen et al., 2010; Matsunaga et al., 1995).The cells were first stained with PI and then with DAPI. Bacterial cellswere grown until they reached the mid-logarithmic phase and then they(˜2×10⁶ cells) were incubated with the lipidated γ-AApeptides at theconcentration of 2×MIC (10 μg/ml) for 2 h. Then the cells were pelletedby centrifugation at 3000 g for 15 min in an Eppendorf microcentrifuge.The supernatant was then decanted and the cells were washed with 1×PBSseveral times and then incubated with PI (5 μg/ml) in the dark for 15min at 0° C. The excess PI was removed by washing the cells with 1×PBSseveral times. Then the cells were incubated with DAPI (10 μg/ml inwater) for 15 mins in the dark at 0° C. The DAPI solution was removedand cells were washed with 1×PBS several times. Controls were performedfollowing the exact same procedure for bacteria without the addition ofγ-AApeptides. The bacterial cells were then examined by using the ZeissAxio Imager Z1 optical microscope with an oil-immersion objective (100×)(Williams et al., 1998).

Parts a1 and b1 of FIG. 13 show that, without treatment by Sequence 13,both E. coli and B. subtilis stained with DAPI but not PI, indicatingthat their membranes are intact. When cells were treated with Sequence13 for 2 h, however, both E. coli and B. subtilis strongly stained withboth DAPI (parts a3 and b3 of FIG. 13) and PI (parts a4 and b4 of FIG.13), demonstrating membrane disruption. The aggregation of dead cells isalso observed because of the loss of membrane potential, which isconsistent with previous studies on antimicrobial peptide amphiphiles(Chen et al., 2010). Similar membrane disruption was also seen with thetreatment of Sequence 6 (FIG. 14).

6. Lipid Depolarization (Wu et al., 1999; Choi et al., 2009; Friedrichet al., 2000)

The antimicrobial mechanism of membrane disruption for lipidatedγ-AApeptides was further investigated using the membrane depolarizationassay (DiSC₃-5 assay described above). S. aureus (ATCC 33592) cells weregrown in Luria broth and Trypticase soy broth medium respectively to amid-logarithmic phase (OD600=0.5-0.6). The bacterial cells were thencollected by centrifugation at 3000 rpm for 10 min and then washed oncewith buffer (5 mM HEPES and 5 mM glucose, pH 7.2). The cells werere-suspended to OD600=0.05 with 100 mM KCl, 2 μM DiSC3-5.5 mM HEPES and5 mM glucose and were incubated for 30 min at 37° C. for maximal dyeuptake and fluorescence self-quenching. This bacterial suspension (50μL) and 50 μL of lipidated γ-AApeptide stock solutions or control drugsolution were added to white flat-bottomed polypropylene 96-well plates(Costar). The fluorescence reading was recorded every 2 min for 30 minusing the microplate reader (Biotek) at an excitation wavelength of 622nm and an emission wavelength of 670 nm. Valinomycin (finalconcentration 250 μg/mL) was used as a positive control, and the blankwith only cells and dye was used as the background.

MRSA, a clinically relevant and widely drug resistant bacterialpathogen, was selected for this study using the membranepotential-sensitive dye DiSC3 to test membrane integrity (Choi et al.,2009). Consistent with previous reports (Choi et al., 2009), ouranalysis shows that the concentrations of lipidated γ-AApeptidesrequired for complete depolarization are much higher than their MICs,and there is no perfect relationship between the MIC and the capabilityfor depolarization (FIG. 15). However, the treatment of MRSA withlipidated γ-AApeptides led to dramatically increased fluorescence, whichwas maximal after 10 min (FIG. 15). Meanwhile, there is a general trendthat lipidated γ-AApeptides with higher MICs require higherconcentrations to cause the same degree of depolarization than thosewith lower MICs. These data further suggest that lipidated γ-AApeptideskill bacteria via membrane disruption.

7. Drug Resistance Study

To investigate the potential of lipidated γ-AApeptides to select fordrug-resistant isolates, methicillin-resistant S. aureus was seriallypassaged on half-MIC concentrations of Sequence 6, with new MIC valuesdetermined every 24 h. Sequence 6 was chosen as a representativesequence as a result of its broad spectrum of activity against testmicroorganisms. As a positive control, parallel cultures were exposed to2-fold dilutions of the antibiotic norfloxacin (FIG. 16) (Choi et al.,2009).

The initial MIC of Sequence 6 and control antibiotic norfloxacin againstS. aureus was obtained as described above. Bacteria from duplicate wellsat the concentration of one-half MIC were then used to prepare thebacterial dilution (approximately 106 CFU/ml) for the next experiment.These bacterial suspensions were then incubated with Sequence 6 andnorfloxacin respectively. After incubation at 37° C. for 24 h, the newMIC was determined. The experiment was repeated each day for 17passages.

While there are almost no changes in the MIC for Sequence 6 after 17days with 17 passages, an increase in MIC for norfloxacin was foundafter just three passages, with a more than 20-fold increase in MICobserved after 17 days. These findings further support that lipidatedγ-AApeptides do not readily permit the development of drug resistance.

8. MTT Assay

To further assess the potential of γ-AApeptide amphiphiles as novelantibiotics, we also evaluated the toxicity of Sequence 6 towardmammalian cells using an MTT assay (FIG. 17). N2a APP cells were used toaccess the cell viability after treatment with Sequence 6. Typically,stock concentration of Sequence 6 (1 mg/mL) was diluted in medium in a96-well plate to make different concentrations and then incubated at 37°C. In another 96-well plate, N2a APP cells were seeded to 1×10⁴cells/well, each of which contained 100 μL of medium. After incubationfor 12 h, an amount of 100 μL of different concentrations of Sequence 6was added and the plate was incubated for another 36 h. At 1 h beforetime is due, MTT reagent (Roche) was incubated at 37° C. degree waterbath. The medium in the 96-well plate was removed and washed with freshmedium once, followed by adding 110 μL of MTT reagent, and thenincubated for another 4 h, after which 100 μL of prewarmedsolubilization solution was added. The plate was then incubated at 37°C. for 12 h before absorbance at 550 nm was read. Percentage of cellviability was calculated based on the following equation:Cell viability %=(A/A _(control))×100

At concentrations up to 50 μg/mL of Sequence 6, almost no toxicity wasobserved, while at 100 μg/mL of Sequence 6, only around 50% of N2a APPcell viability was compromised. These results show that selectivity is10- to 50-fold lower than for bacteria, further demonstrating thefeasibility of lipidated γ-AApeptides for use as antimicrobialtherapeutics.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) of any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated within the scope of the invention without limitationthereto.

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We claim:
 1. A γ-AApeptide prepared by a process comprising the stepsof: reacting an Fmoc protected amino aldehyde comprising:

wherein R₁ is a straight or branched chain C₁ to C ₁₀ alkyl group,—CH₂—CH₂—S—CH₃; a —(CH₂)₁₋₅-aryl group, or an —(CH₂)₁₋₅-heteroarylgroup, and wherein the alkyl group, the aryl group or the heteroarylgroup can be substituted or unsubstituted, with glycine benzyl estercomprising:

to form a secondary amine comprising:

reacting

with a substituted or unsubstituted aryl, substituted or unsubstituted5-membered heterocyclic ring of which one to four member(s) may beheteroatoms, γ-Boc-amino butyric acid, di-Boc-guanidinopropionic acid,mono-allyl succinate or substituted or unsubstituted alkanoic acidfollowed by hydrogenation to form γ-AApeptide building blockscomprising:

wherein R₂ is substituted or unsubstituted aryl, substituted orunsubstituted 5-membered heterocyclic ring of which one to fourmember(s) may be heteroatoms, amino-propyl, allyl-propyl, or substitutedor unsubstituted alkyl; attaching a first γ-AApeptide building block toa solid support; coupling the first γ-AApeptide building block with asecond γ-AApeptide building block; repeating the coupling step to formthe γ-AApeptide attached to the solid support; and cleaving theγ-AApeptide attached to the solid support from the solid support to formthe γ-AApeptide.
 2. The γ-AApeptide prepared by the process of claim 1,wherein the solid support is a Rink amide resin or a Knorr resin.
 3. Theγ-AApeptide prepared by the process of claim 1, wherein R₁ is a methyl,ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, s-butyl ort-butyl group.
 4. The γ-AApeptide prepared by the process of claim 1,wherein the repeating step is performed to couple between 5 and 50γ-AApeptide building blocks.
 5. The γ-AApeptide prepared by the processof claim 1, wherein the step of reacting an Fmoc protected aminoaldehyde with glycine benzyl ester further comprises sodiumcyanoborohydride.
 6. The γ-AApeptide prepared by the process of claim 1,wherein the step of reacting

with the substituted or unsubstituted aryl, substituted or unsubstituted5-membered heterocyclic ring of which one to four member(s) may beheteroatoms, γ-Boc-amino butyric acid, di-Boc-guanidinopropionic acid,mono-allyl succinate or substituted or unsubstituted alkanoic acidfurther comprises diisopropylcarbodiimide,3-4-dihydro-3-hydroxy-4-oxo-1-2-3-benzotriazine, and dimethylformamide.7. The γ-AApeptide prepared by the process of claim 1, whereinhydrogenation to form γ-AApeptide building blocks comprises a palladiumon carbon catalyst and molecular hydrogen.
 8. The γ-AApeptide preparedby the process of claim 1, wherein attaching a first γ-AApeptidebuilding block to a solid support further comprisesN,N′-Diisopropylearbodiimide and oxohydroxybenzotriazole.
 9. Theγ-AApeptide prepared by the process of claim 1, wherein coupling thefirst γ-AApeptide building block with a second γ-AApeptide buildingblock further comprises N,N′-Diisopropylcarbodiimide andoxohydroxybenzotriazole.
 10. The γ-AApeptide prepared by the process ofclaim 1, wherein coupling the first γ-AApeptide building block with asecond γ-AApeptide building block further comprisesbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate. 11.The γ-AApeptide prepared by the process of claim 1, wherein coupling thefirst γ-AApeptide building block with a second γ-AApeptide buildingblock further comprises2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateand hydroxybenzotriazole.
 12. The γ-AApeptide prepared by the process ofclaim 1, wherein cleaving the γ-AApeptide attached to the solid supportfrom the solid support to form the γ-AApeptide further comprisestrifluoroacetic acid, methylene chloride, and triisopropylsilane. 13.The γ-AApeptide prepared by the process of claim 1, wherein theγ-AApeptide is a cyclic γ-AApeptide.
 14. The γ-AApeptide prepared by theprocess of claim 1, the process further comprising the step of purifyingthe γ-AApeptide by high performance liquid chromatography.
 15. Theγ-AApeptide prepared by the process of claim 11, the process furthercomprising lyophizing the γ-AApeptide.
 16. The γ-AApeptide prepared bythe process of claim 1, the process further comprising: lyophizing theγ-AApeptide.
 17. The γ-AApeptide prepared by the process of claim 1,wherein the substituted or unsubstituted alkanoic acid is ethanoic acidor 4-methyl pentanoic acid.
 18. The γ-AApeptide prepared by the processof claim 1, wherein the substituted or unsubstituted 5-memberedheterocyclic ring is selected from furanyl, thienyl, pyrrolyl, N-alkylpyrrolyl, or imidazole.
 19. The γ-AApeptide prepared by the process ofclaim 1, wherein the substituted or unsubstituted aryl is a substitutedor unsubstituted hydrocinnamic acid.
 20. The γ-AApeptide prepared by theprocess of claim 19, wherein the substituted or unsubstitutedhydrocinnamic acid is α-cyano-4-hydroxy-cinnamic acid.